Oil-in-water emulsions that contain nucleic acids

ABSTRACT

This invention generally relates to cationic oil-in-water emulsions that can be used to deliver nucleic acid molecules, such as an RNA molecule. The emulsion particles comprise an oil core and a cationic lipid. The emulsion particles have an average diameter of about 80 nm to about 180 nm, and the emulsion have an N/P ratio of at least 1.1:1.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 3, 2014, isnamed PAT054719.txt and is 32,549 bytes in size.

BACKGROUND OF THE INVENTION

Nucleic acid therapeutics have promise for treating diseases rangingfrom inherited disorders to acquired conditions such as cancer,infectious disorders (AIDS), heart disease, arthritis, andneurodegenerative disorders (e.g., Parkinson's and Alzheimer's). Notonly can functional genes be delivered to repair a genetic deficiency orinduce expression of exogenous gene products, but nucleic acid can alsobe delivered to inhibit endogenous gene expression to provide atherapeutic effect. Inhibition of gene expression can be mediated by,e.g., antisense oligonucleotides, double-stranded RNAs (e.g., siRNAs,miRNAs), or ribozymes.

A key step for such therapy is to deliver nucleic acid molecules intocells in vivo. However, in vivo delivery of nucleic acid molecules, inparticular RNA molecules, faces a number of technical hurdles. First,due to cellular and serum nucleases, the half life of RNA injected invivo is only about 70 seconds (see, e.g., Kurreck, Eur. J. Bioch.270:1628-44 (2003)). Efforts have been made to increase stability ofinjected RNA by the use of chemical modifications; however, there areseveral instances where chemical alterations led to increased cytotoxiceffects or loss of or decreased function. In one specific example, cellswere intolerant to doses of an RNAi duplex in which every secondphosphate was replaced by phosphorothioate (Harborth, et al, AntisenseNucleic Acid Drug Rev. 13(2): 83-105 (2003)). As such, there is a needto develop delivery systems that can deliver sufficient amounts ofnucleic acid molecules (in particular RNA molecules) in vivo to elicit atherapeutic response, but that are not toxic to the host.

Nucleic acid based vaccines are an attractive approach to vaccination.For example, intramuscular (IM) immunization of plasmid DNA encoding forantigen can induce cellular and humoral immune responses and protectagainst challenge. DNA vaccines offer certain advantages overtraditional vaccines using protein antigens, or attenuated pathogens.For example, as compared to protein vaccines, DNA vaccines can be moreeffective in producing a properly folded antigen in its nativeconformation, and in generating a cellular immune response. DNA vaccinesalso do not have some of the safety problems associated with killed orattenuated pathogens. For example, a killed viral preparation maycontain residual live viruses, and an attenuated virus may mutate andrevert to a pathogenic phenotype.

Another limitation of nucleic acid based vaccines is that large doses ofnucleic acid are generally required to obtain potent immune responses innon-human primates and humans. Therefore, delivery systems and adjuvantsare required to enhance the potency of nucleic acid based vaccines.Various methods have been developed for introducing nucleic acidmolecules into cells, such as calcium phosphate transfection, polyprenetransfection, protoplast fusion, electroporation, microinjection andlipofection.

Cationic lipids have been widely formulated as liposomes to delivergenes into cells. However, even a small amount of serum (˜10%) candramatically reduce the transfection activity of liposome/DNA complexesbecause serum contains anionic materials. Recently, cationic lipidemulsion was developed to deliver DNA molecules into cells. See, e.g.,Kim, et al., International Journal of Pharmaceutics, 295, 35-45 (2005).

U.S. Pat. Nos. 6,753,015 and 6,855,492 describe a method of deliveringnucleic acid molecules to a vertebrate subject using cationicmicroparticles. The microparticles comprise a polymer, such as apoly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester, a polyanhydride, and the like, and are formed usingcationic surfactants. Nucleic acid molecules are adsorbed on thesurfaces of the microparticles.

Kim et al. (Pharmaceutical Research, vol. 18, pages 54-60, 2001) andChung et al. (Journal of Controlled Release, volume 71, pages 339-350,2001) describe various oil-in-water emulsion formulations that are usedto enhance in vitro and in vivo transfection efficiency of DNAmolecules.

Ott et al. (Journal of Controlled Release, volume 79, pages 1-5, 2002)describes an approach involving a cationic sub-micron emulsion as adelivery system/adjuvant for DNA. The sub-micron emulsion approach isbased on MF59, a potent squalene in water adjuvant which has beenmanufactured at large scale and has been used in a commercially approvedproduct (Fluad®). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) wasused to facilitate intracellular delivery of plasmid DNA.

Although DNA-based vaccines hold great promise for prevention andtreatment of diseases, general concerns have been raised regarding theirsafety. The introduced DNA molecules could potentially integrate intothe host genome or, due to their distribution to various tissues, couldlead to undesirable sustained expression of antigens. In addition,certain DNA viruses have also been used as a vehicle to deliver DNAmolecules. Because of their infectious properties, such viruses achievea very high transfection rate. The viruses used are genetically modifiedin such a manner that no functional infectious particles are formed inthe transfected cell. Despite these precautions, however, it is notpossible to rule out the risk of uncontrolled propagation of theintroduced gene and viral genes, for example due to potentialrecombination events. This also entails the risk of the DNA beinginserted into an intact gene of the host cell's genome by e.g.recombination, with the consequence that this gene may be mutated andthus completely or partially inactivated or may give rise tomisinformation. In other words, synthesis of a gene product which isvital to the cell may be completely suppressed or, alternatively, amodified or incorrect gene product is expressed. In addition, it isgenerally difficult to scale up the manufacture and purification ofclinical-grade viral vectors.

One particular risk occurs if the DNA is integrated into a gene which isinvolved in the regulation of cell growth. In this case, the host cellmay become degenerate and lead to cancer or tumor formation.Furthermore, if the DNA introduced into the cell is to be expressed, itis necessary for the corresponding DNA vehicle to contain a strongpromoter, such as the viral CMV promoter. The integration of suchpromoters into the genome of the treated cell may result in unwantedalterations of the regulation of gene expression in the cell. Anotherrisk of using DNA as an agent to induce an immune response (e.g. as avaccine) is the induction of pathogenic anti-DNA antibodies in thepatient into whom the foreign DNA has been introduced, so bringing aboutan undesirable immune response.

RNA molecules encoding an antigen or a derivative thereof may also beused as vaccines. RNA vaccines offer certain advantages as compared toDNA vaccines. First, RNA cannot integrate into the host genome thusabolishing the risk of malignancies. Second, due to the rapiddegradation of RNA, expression of the foreign transgene is oftenshort-lived, avoiding uncontrolled long term expression of the antigen.Third, RNA molecules only need to be delivered to the cytoplasm toexpress the encoded antigen, whereas DNA molecules must permeate throughthe nuclear membrane.

Nonetheless, compared with DNA-based vaccines, relatively minorattention has been given to RNA-based vaccines. RNAs andoligonucleotides are hydrophilic, negatively charged molecules that arehighly susceptible to degradation by nucleases when administered as atherapeutic or vaccine. Additionally, RNAs and oligonucleotides are notactively transported into cells. See, e.g., Vajdy, M., et al., Mucosaladjuvants and delivery systemsfor protein-, DNA-and RNA-based vaccines,Immunol Cell Biol, 2004. 82(6): p. 617-27.

Ying et al. (Nature Medicine, vol. 5, pages 823-827, 1999) describes aself-replicating RNA vaccine in which naked RNA encoding β-galactosidasewas delivered and the induction of CD8+ cells was reported.

Montana et al. (Bioconjugate Chem. 2007, 18, pages 302-308) describesusing cationic solid-lipid nanoparticles as RNA carriers for genetransfer. It was shown that solid-lipid nanoparticles protected the RNAmolecule from degradation, and the expression of reporter protein(fluorescein) was detected after microinjecting the RNA-particle complexinto sea urchin eggs.

WO 2010/009277 discloses Nano Lipid Peptide Particles (NLPPs) comprising(a) an amphipathic peptide, (b) a lipid, and (c) at least oneimmunogenic species. In certain embodiments, the NLPPs also incorporatea positively charged “capturing agent,” such as a cationic lipid. Thecapturing agent is used to anchor a negatively charged immunogenicspecies (e.g., a DNA molecule or an RNA molecule). Preparation of NLPPrequires amphipathic peptides, which are used to solubilize the lipidcomponent and to form nano-particles.

Therefore, there is a need to provide delivery systems for nucleic acidmolecules. The delivery systems are useful for nucleic acid-basedvaccines, in particular RNA-based vaccines.

SUMMARY OF THE INVENTION

This invention generally relates to cationic oil-in-water emulsions inwhich a nucleic acid molecule is complexed to the emulsion particles.The emulsions can be used to deliver nucleic acide molecules, such as anRNA molecule to cells. The emulsion particles comprise an oil core and acationic lipid. The cationic lipid can interact with the negativelycharged molecule thereby anchoring the molecule to the emulsionparticles. The emulsion particles have an average diameter of about 80nm to about 180 nm, and the emulsion have an N/P ratio of at least 4:1.

In some embodiments, the emulsion particles have an average diameter ofabout 80 nm to about 180 nm, and the emulsion have an N/P ratio of atleast 1.1:1, at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1,or at least 3.5:1.

The invention provides an immunogenic cationic oil-in-water emulsioncomprising emulsion particles that contain an oil core and a cationiclipid, and a nucleic acid molecule that is complexed to the emulsionparticles, and wherein the average diameter of the emulsion particles isfrom about 80 nm to about 180 nm and the N/P of the emulsion is at least4:1; with the proviso that the nucleic acid molecule does not encodesecreted alkaline phosphatase, and the further proviso that the nucleicacid molecule is not an RNA encoded by the plasmid A317, the sequence ofwhich is shown in FIG. 7A of U.S. Patent Application No. 61/361,892. Insome embodiments, the invention includes the further proviso that thenucleic acid molecule does not encode an RSV F protein antigen, and/orthe further proviso that the nucleic acid molecule does not encode anRSV protein. Preferably, the nucleic acid molecule encodes an antigen,and is an RNA molecule, such as a self-replicating RNA.

The immunogenic cationic oil-in-water emulsion can be buffered, (e.g.,with a citrate buffer, a succinate buffer, an acetate buffer) and has apH from about 6.0 to about 8.0, preferably about 6.2 to about 6.8.Optionally, the immunogenic cationic oil-in-water emulsion furthercomprises an inorganic salt, preferably at a concentration no greaterthan 30 mM. Alternatively or in addition, the immunogenic cationicoil-in-water emulsion further comprises a nonionic tonicifying agent,such as a sugar, a sugar alcohol or combinations thereof, and or apolymer, such as a poloxamer, in the aqueous phase. If present, thepolymer can be present in about 0.05% to about 20% (w/v). In someembodiments, immunogenic cationic oil-in-water emulsion is isotonic. Inother embodiments, immunogenic cationic oil-in-water emulsions arehypotonic or hypertonic.

In some embodiments, the oil core of the emulsion parties comprises anoil that is selected from the group consisting of: Castor oil, Coconutoil, Corn oil, Cottonseed oil, Evening primrose oil, Fish oil, Jojobaoil, Lard oil, Linseed oil, Olive oil, Peanut oil, Safflower oil, Sesameoil, Soybean oil, Squalene, Squalane, Sunflower oil, Wheatgerm oil, andcombinations thereof. In particular embodiments, the oil core issqualene. The cationic lipid can be selected from the group consistingof: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), Lipids E0001-E0118,and combinations thereof. In a preferred embodiment, the cationic lipidis DOTAP.

The emulsion particles can further comprise a surfactant, such as anonionic surfactant. Preferably, the surfactant is not a PolyethyleneGlycol (PEG)-lipid. The surfactant can be present in an amount fromabout 0.01% to about 2.5% (w/v). In some embodiments, the surfactant isSPAN85 (Sorbtian Trioleate), Tween 80 (polysorbate 80), or a combinationthereof. In some embodiments, the oil-in-water emulsion contains equalamounts of SPAN85 (Sorbtian Trioleate) and Tween 80 (polysorbate 80),for example 0.5% (w/v) of each particle further comprises a surfactant.

The invention also relates to a method for making immunogenic cationicoil-in-water emulsion comprising emulsion particles that contain an oilcore and a cationic lipid, and a nucleic acid molecule that is complexedto the emulsion particles, and wherein the average diameter of theemulsion particles is from about 80 nm to about 180 nm and the N/P ofthe emulsion is at least 4:1; with the proviso that the nucleic acidmolecule does not encode secreted alkaline phosphatase, and the furtherproviso that the nucleic acid molecule is not an RNA encoded by theplasmid A317, the sequence of which is shown in FIG. 7A of U.S. PatentApplication No. 61/361,892. The method comprises providing (i) acationic oil-in-water emulsions containing emulsion particles thatcontain an oil core and a cationic lipid as described herein, and (ii)an aqueous solution that contains a nucleic acid, and combining (i) and(ii) thereby making said immunogenic cationic oil-in-water emulsion.

The invention also relates to a method for generating an immune responsein a subject, comprising administering to the subject an effectiveamount of an immunogenic cationic oil-in-water emulsion describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the results of an in vivo SEAP assay, using 1 μg of RNAreplicon vA306 complexed with CNE17 at 10:1 N:P ratio. FIG. 1B shows thetotal IgG titers in BALB/c mice at 2wp1 and 2wp2 time points followingimmunization with RNA replicon A317, which encodes RSV F protein,complexed with CNE17. These data show that as particle size increased,SEAP expression decreased, and the immune response to RSV F proteindecreased.

FIGS. 2A-2C show the effects of RNA complexation and different buffercompositions on particle size. FIG. 2A shows that RNA complexationincreased the particle size in CNE 17 emulsion, and that sugar and/orlow salt (NaCl, 10 mM) decreased particle size. The addition of PluronicF127 reduced particle size to precomplexation size. FIG. 2B shows theeffect of citrate buffer on particle size. FIG. 2C shows the effect ofpolymers on particle size.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

This invention generally relates to cationic oil-in-water emulsions inwhich a nucleic acid molecule is complexed to the emulsion particles,the emulsion particles have an average diameter of about 80 nm to about180 nm, and the emulsion have an N/P ratio of at least 4:1. The emulsionparticles comprise an oil core and a cationic lipid. The cationic lipidcan interact with the negatively charged molecule thereby anchoring themolecule to the emulsion particles. The emulsions can be used to delivernucleic acide molecules, such as an RNA molecule to cells.

The present invention is based on the discovery that an immune responsethat is raised against an antigen enconded by RNA, when a cationicoil-in-water emulsion that contains an RNA molecule encoding the antigenis administered to an individual, is affected by properties of theemulsion, including the average diameter of the emulsion particles andthe amount of RNA that is complexed to the emulsion particles (i.e., N/Pratio). The inventors determined that the average diameter of emulsionparticles increases when RNA molecules are complexed to the emulsion,and that large diameter particles result in lower neutralizing titers.Emulsions in which the average particle diameter is between about 80 nmand about 180 nm provide good neutralizing titers. Even within thisrange, emulsions with average particle sizes at the high end of therange are less preferred, and an average particle size of about 100 nmis particularly preferred. Also preferred is an average particlediameter between about 100 nm and about 130 nm. The inventorssurprisingly discovered that the immune response does not have a directlinear relationship to the amount of RNA that is complexed to theemulsion. An N/P ratio of 4:1 produces a good immune response to theencoded antigen, but higher N/P ratios do not necessarily result inhigher neutralizing titers.

The average diameter of cationic oil-in-water emulsion particlesincreases when RNA molecules are complexed to the emulsion. This effectcan be reduced by the inclusion of inorganic salts in the emulsion.However, inorganic salts can inhibit the binding of nucleic acidmolecules to the emulsion particles, therefore, the inorganic salt(s)should be present in only low concentrations (e.g., no more than about30 mM). If desired, hydrophilic polymers can be used to reduce theparticle size increase caused by nucleic acid complexation. Again,hydrophilic polymers can inhibit binding of nucleic acid molecules tothe emulsion particles, therefore, hydrophilic polymer(s) should bepresent in only low concentrations (e.g., about 0.05% to about 20%(w/v)).

Cationic oil-in-water emulsions that contain an RNA molecule encodingthe antigen can be administered to an individual to induce an immuneresponse to the encoded antigen. When the cationic oil-in-wateremulsions are used for this purpose, it is preferred that they areisotonic. However, because inorganic salts can inhibit the binding ofnucleic acid molecules to the emulsion particles, tonicity is preferablyadjusted with a nonionic tonicifying agent. In preferred embodiments,the cationic oil-in-water emulsions that contain an RNA molecule areadjusted to 250 mOsm/kg water to about 320 mOsm/kg using a sugar, sugaralcohol or combination thereof.

2. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise.

The term “about” as used here, refers to +/−10% of a value.

The term “surfactant” is a term of art and generally refers to anymolecule having both a hydrophilic group (e.g., a polar group), whichenergetically prefers solvation by water, and a hydrophobic group whichis not well solvated by water. The term “nonionic surfactant” is a knownterm in the art and generally refers to a surfactant molecule whosehydrophilic group (e.g., polar group) is not electrostatically charged.

The term “polymer” refers to a molecule consisting of individualchemical moieties, which may be the same or different, that are joinedtogether. As used herein, the term “polymer” refers to individualchemical moieties that are joined end-to-end to form a linear molecule,as well as individual chemical moieties joined together in the form of abranched (e.g., a “multi-arm” or “star-shaped”) structure. Exemplarypolymers include, e.g., poloxamers. Poloxamers are nonionic triblockcopolymers having a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)).

A “buffer” refers to an aqueous solution that resists changes in the pHof the solution.

As used herein, “nucleotide analog” or “modified nucleotide” refers to anucleotide that contains one or more chemical modifications (e.g.,substitutions) in or on the nitrogenous base of the nucleoside (e.g.,cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)). Anucleotide analog can contain further chemical modifications in or onthe sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modifiedribose, modified deoxyribose, six-membered sugar analog, or open-chainsugar analog), or the phosphate.

As use herein, “saccharide” encompasses monosaccharides,oligosaccharides, or polysaccharides in straight chain or ring forms, ora combination thereof to form a saccharide chain. Oligosaccharides aresaccharides having two or more monosaccharide residues. Examples ofsaccharides include glucose, maltose, maltotriose, maltotetraose,sucrose and trehalose.

The terms “self-replicating RNA,” “RNA replicon” or “RNA vector” is aterm of art and generally refer to an RNA molecule which is capable ofdirecting its own amplification or self-replication in vivo, typicallywithin a target cell. The RNA replicon is used directly, without therequirement for introduction of DNA into a cell and transport to thenucleus where transcription would occur. By using the RNA vector fordirect delivery into the cytoplasm of the host cell, autonomousreplication and translation of the heterologous nucleic acid sequenceoccurs efficiently. An alphavirus-derived self-replicating RNA maycontain the following elements in sequential order: 5′ viral sequencesrequired in cis for replication (also referred to as 5′ CSE, inbackground), sequences which, when expressed, code for biologicallyactive alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4),3′ viral sequences required in cis for replication (also referred to as3′ CSE, in background), and a polyadenylate tract. Thealphavirus-derived self-replicating RNA may also contain a viralsubgenomic “junction region” promoter, sequences from one or morestructural protein genes or portions thereof, extraneous nucleic acidmolecule(s) which are of a size sufficient to allow production ofrecombinant alphavirus particles, as well as heterologous sequence(s) tobe expressed.

The term “adjuvant” refers to any substance that assists or modifies theaction of a pharmaceutical, including but not limited to immunologicaladjuvants, which increase and/or diversify the immune response to anantigen. Hence, immunological adjuvants include compounds that arecapable of potentiating an immune response to antigens. Immunologicaladjuvants can potentiate humoral and/or cellular immunity. Substancesthat stimulate an innate immune response are included within thedefinition of immunological adjuvants herein. Immunological adjuvantsmay also be referred to as “immunopotentiators.”

As used herein, an “antigen” refers to a molecule containing one or moreepitopes (e.g., linear, conformational or both). As used herein, an“epitope” is that portion of given species (e.g., an antigenic moleculeor antigenic complex) that determines its immunological specificity. Anepitope is within the scope of the present definition of antigen. Theterm “antigen” as used herein includes subunit antigens, i.e., antigenswhich are separate and discrete from a whole organism with which theantigen is associated in nature.

An “immunological response” or “immune response” is the development in asubject of a humoral and/or a cellular immune response to an antigen oran immunological adjuvant.

Immune responses include innate and adaptive immune responses. Innateimmune responses are fast-acting responses that provide a first line ofdefense for the immune system. In contrast, adaptive immunity usesselection and clonal expansion of immune cells having somaticallyrearranged receptor genes (e.g., T- and B-cell receptors) that recognizeantigens from a given pathogen or disorder (e.g., a tumor), therebyproviding specificity and immunological memory. Innate immune responses,among their many effects, lead to a rapid burst of inflammatorycytokines and activation of antigen-presenting cells (APCs) such asmacrophages and dendritic cells. To distinguish pathogens fromself-components, the innate immune system uses a variety of relativelyinvariable receptors that detect signatures from pathogens, known aspathogen-associated molecular patterns, or PAMPs. The addition ofmicrobial components to experimental vaccines is known to lead to thedevelopment of robust and durable adaptive immune responses. Themechanism behind this potentiation of the immune responses has beenreported to involve pattern-recognition receptors (PRRs), which aredifferentially expressed on a variety of immune cells, includingneutrophils, macrophages, dendritic cells, natural killer cells, B cellsand some nonimmune cells such as epithelial and endothelial cells.Engagement of PRRs leads to the activation of some of these cells andtheir secretion of cytokines and chemokines, as well as maturation andmigration of other cells. In tandem, this creates an inflammatoryenvironment that leads to the establishment of the adaptive immuneresponse. PRRs include nonphagocytic receptors, such as Toll-likereceptors (TLRs) and nucleotide-binding oligomerization domain (NOD)proteins, and receptors that induce phagocytosis, such as scavengerreceptors, mannose receptors and β-glucan receptors. Reported TLRs(along with examples of some reported ligands, which may be used asimmunogenic molecule in various embodiments of the invention) includethe following: TLR1 (bacterial lipoproteins from Mycobacteria,Neisseria), TLR2 (zymosan yeast particles, peptidoglycan, lipoproteins,lipopeptides, glycolipids, lipopolysaccharide), TLR3 (viraldouble-stranded RNA, poly:IC), TLR4 (bacterial lipopolysaccharides,plant product taxol), TLR5 (bacterial flagellins), TLR6 (yeast zymosanparticles, lipotechoic acid, lipopeptides from mycoplasma), TLR7(single-stranded RNA, imiquimod, resimiquimod, and other syntheticcompounds such as loxoribine and bropirimine), TLR8 (single-strandedRNA, resimiquimod) and TLR9 (CpG oligonucleotides), among others.Dendritic cells are recognized as some of the most important cell typesfor initiating the priming of naive CD4⁺ helper T (T_(H)) cells and forinducing CD8⁺ T cell differentiation into killer cells. TLR signalinghas been reported to play an important role in determining the qualityof these helper T cell responses, for instance, with the nature of theTLR signal determining the specific type of T_(H) response that isobserved (e.g., T_(H)1 versus T_(H)2 response). A combination ofantibody (humoral) and cellular immunity are produced as part of aT_(H)I-type response, whereas a T_(H)2-type response is predominantly anantibody response. Various TLR ligands such as CpG DNA (TLR9) andimidazoquinolines (TLR7, TLR8) have been documented to stimulatecytokine production from immune cells in vitro. The imidazoquinolinesare the first small, drug-like compounds shown to be TLR agonists. Forfurther information, see, e.g., A. Pashine, N. M. Valiante and J. B.Ulmer, Nature Medicine 11, S63-S68 (2005), K. S. Rosenthal and D. H.Zimmerman, Clinical and Vaccine Immunology, 13(8), 821-829 (2006), andthe references cited therein.

For purposes of the present invention, a humoral immune response refersto an immune response mediated by antibody molecules, while a cellularimmune response is one mediated by T-lymphocytes and/or other whiteblood cells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (CTLs). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4⁺ and CD8⁺ T-cells.

A composition such as an immunogenic composition or a vaccine thatelicits a cellular immune response may thus serve to sensitize avertebrate subject by the presentation of antigen in association withMHC molecules at the cell surface. The cell-mediated immune response isdirected at, or near, cells presenting antigen at their surface. Inaddition, antigen-specific T-lymphocytes can be generated to allow forthe future protection of an immunized host. The ability of a particularantigen or composition to stimulate a cell-mediated immunologicalresponse may be determined by a number of assays known in the art, suchas by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, by assaying for T-lymphocytes specific for the antigen in asensitized subject, or by measurement of cytokine production by T cellsin response to restimulation with antigen. Such assays are well known inthe art. See, e.g., Erickson et al. (1993) J. Immunol 151:4189-4199; Doeet al. (1994) Eur. J. Immunol 24:2369-2376. Thus, an immunologicalresponse as used herein may be one which stimulates the production ofCTLs and/or the production or activation of helper T-cells. The antigenof interest may also elicit an antibody-mediated immune response. Hence,an immunological response may include, for example, one or more of thefollowing effects among others: the production of antibodies by, forexample, B-cells; and/or the activation of suppressor T-cells and/or γδT-cells directed specifically to an antigen or antigens present in thecomposition or vaccine of interest. These responses may serve, forexample, to neutralize infectivity, and/or mediate antibody-complement,or antibody dependent cell cytotoxicity (ADCC) to provide protection toan immunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

Compositions in accordance with the present invention display “enhancedimmunogenicity” for a given antigen when they possess a greater capacityto elicit an immune response than the immune response elicited by anequivalent amount of the antigen in a differing composition (e.g.,wherein the antigen is administered as a soluble protein). Thus, acomposition may display enhanced immunogenicity, for example, becausethe composition generates a stronger immune response, or because a lowerdose or fewer doses of antigen is necessary to achieve an immuneresponse in the subject to which it is administered. Such enhancedimmunogenicity can be determined, for example, by administering acomposition of the invention and an antigen control to animals andcomparing assay results of the two.

3. Cationic Oil-in-Water Emulsions

The cationic oil-in-water emulsions disclosed herein are generallydescribed in the manner that is conventional in the art, byconcentrations of components that are used to prepare the emulsions. Itis understood in the art that during the process of producing emulsions,including sterilization and other downstream processes, small amounts ofoil (e.g., squalene), cationic lipid (e.g., DOTAP), or other componentsmay be lost, and the actual concentrations of these components in thefinal product (e.g., a packaged, sterilized emulsion that is ready foradministration) might be slightly lower than starting amounts, sometimesby up to about 10% or by up to about 20%.

The cationic oil-in-water emulsion particles comprise an oil core and acationic lipid. The cationic lipid can interact with the nucleic acidmolecule, for example through electrostatic forces andhydrophobic/hydrophilic interactions, thereby anchoring the nucleic acidmolecule to the emulsion particles. The cationic emulsions describedherein are particularly suitable for delivering a nucleic acid molecule,such as an RNA molecule encoding an antigen or small interfering RNA tocells in vivo. For example, the cationic emulsions described hereinprovide advantages for delivering RNA that encode antigens, includingself-replicating RNAs, as vaccines.

The particles of the oil-in-water emulsions likely resemble a micellewith a central core of oil. The oil core is coated with the cationiclipid, which disperses the oil droplet in the aqueous (continuous) phaseas micelle-like droplets. One or more optional components may be presentin the emulsion, such as surfactants and/or phospholipids as describedbelow. For example, one or more surfactants may be used to promoteparticle formation and/or to stabilize the emulsion particles. In thatcase, the oil core is coated with the cationic lipid as well as thesurfactant(s) to form micelle-like droplets. Similarly, one or morelipids (e.g., neutral lipids, glycol-lipids or phospholipids) may alsobe present on the surface of the emulsion particles, if such lipids areused as emulsifiers to disperse the oil droplets.

The particles of the oil-in-water emulsions have an average diameter(i.e., average number diameter) of about 80 nm to about 180 nm, fromabout 80 nm to 150 nm, from about 80 nm to 130 nm, or from about 80 nmto 120 nm. Particularly preferred average particle diameter is about 100nm.

The size of the emulsion particles can be varied by changing the ratioof surfactant to oil (increasing the ratio decreases droplet size),operating pressure of homogenization (increasing operating pressure ofhomogenization typically reduces droplet size), temperature (increasingtemperature decreases droplet size), changing the type of oil,increasing the number of passes through the microfluidizer, and otherprocess parameters, as described herein. Inclusion of certain types ofbuffers in the aqueous phase may also affect the particle size.

The emulsion particles described herein can be complexed with a nucleicacid molecule, as described in further detail herein. In general, acationic oil-in-water emulsion is combined with an aqueous solution thatcontains one or more species of nucleic acid molecules to form theemulsion that contains a nucleic acid molecule that is complexed to theemulsion particles. The aqueous solution that contains the nucleic acidmolecule(s) contains a concentration of nucleic acids that will resultin a complexed emulsion that has an N/P ratio of at least 4:1, forexample from 4:1 to 20:1 or from 4:1 to 15:1. This can easily beaccomplished because the the amount of nitrogen (N) in the emulsion canbe quantified using any suitable method, such as the HPLC method used toquantify DOTAP described herein. Then, an aqueous solution of nucleicacid molecules can be prepared that contains an amount of nucleic acidsufficient to provide the amount of phosphates (P) needed to achieve thedesired N/P ratio.

An exemplary cationic emulsion of the invention is CNE17. The oil coreof CNE17 is squalene (at 4.3% w/v) and the cationic lipid is DOTAP (at1.4 mg/mL). CNE17 also includes the surfactants SPAN85 ((sorbtiantrioleate) at 0.5% v/v) and Tween 80 ((polysorbate 80;polyoxyethylenesorbitan monooleate) at 0.5% v/v). Thus, the particles ofCNE17 comprise a squalene core coated with SPAN85, Tween80, and DOTAP.RNA molecules were shown to complex with CNE17 particles efficiently at4:1 N/P ratio and 10:1 N/P ratio. Another exemplary cationic emulsion ofthe invention is referred herein as “CMF32.” The oil of CMF32 issqualene (at 4.3% w/v) and the cationic lipid is DOTAP (at 3.2 mg/mL).CMF32 also includes the surfactants SPAN85 (sorbitan trioleate at 0.5%v/v) and Tween 80 (polysorbate 80; polyoxyethylenesorbitan monooleate;at 0.5% v/v). Thus, emulsion particles of CMF32 comprise squalene,SPAN85, Tween80, and DOTAP. RNA molecules were shown to complex withCMF32 particles efficiently at 4:1, 6:1, 8:1, 10:1, 12:1, and 14:1 N/Pratios. Other exemplary cationic emulsions include, e.g., the emulsionsreferred to herein as “CMF34” (4.3% w/v squalene, 0.5% Tween 80, 0.5%SPAN85, and 4.4 mg/mL DOTAP), “CMF35” (4.3% w/v squalene, 0.5% Tween 80,0.5% SPAN85, 5.0 mg/mL DOTAP), and other emulsions described herein.

A particular cationic oil-in-water emulsion of the invention comprisesDOTAP and squalene at concentrations of 2.1 mg/ml to 2.84 mg/ml(preferably 2.23 mg/ml to 2.71 mg/ml), and 30.92 mg/ml to 41.92 mg/ml(preferably 32.82 mg/ml to about 40.02 mg/ml), respectively, and furthercomprise equal amounts of SPAN85 and Tween80 (e.g., about 0.5% each).Another particular cationic oil-in-water emulsion of the inventioncomprises DOTAP and squalene at concentrations of 2.78 mg/ml to 3.76mg/ml (preferably 2.94 mg/ml to 3.6 mg/ml), and 18.6 mg/ml to 25.16mg/ml (preferably 19.69 mg/ml to about 24.07 mg/ml), respectively, andfurther comprise equal amounts of SPAN85 and Tween80 (e.g., about 0.5%each). Preferably, the particles of these emulsions have an averagediameter from about 80 nm to about 180 nm.

The individual components of the oil-in-water emulsions of the presentinvention are known in the art, although such compositions have not beencombined in the manner described herein. Accordingly, the individualcomponents, although described below both generally and in some-detailfor preferred embodiments, are well known in the art, and the terms usedherein, such as oil core, surfactant, phospholipids, etc., aresufficiently well known to one skilled in the art without furtherdescription. In addition, while preferred ranges of the amount of theindividual components of the emulsions are provided, the actual ratiosof the components of a particular emulsion may need to be adjusted suchthat emulsion particles of desired size and physical property can beproperly formed. For example, if a particular amount of oil is used(e.g. 5% v/v oil), then, the amount of surfactant should be at levelthat is sufficient to disperse the oil droplet into aqueous phase toform a stable emulsion. The actual amount of surfactant required todisperse the oil droplet into aqueous phase depends on the type ofsurfactant and the type of oil core used for the emulsion; and theamount of oil may also vary according to droplet size (as this changesthe surface area between the two phases). The actual amounts and therelative proportions of the components of a desired emulsion can bereadily determined by a skilled artisan.

A. Oil Core

The particles of the cationic oil-in-water emulsions comprise an oilcore.

The oil preferably is in the liquid phase at 1° C. or above, and isimmiscible to water.

Preferably, the oil is a metabolizable, non-toxic oil; more preferablyone of about 6 to about 30 carbon atoms including, but not limited to,alkanes, alkenes, alkynes, and their corresponding acids and alcohols,the ethers and esters thereof, and mixtures thereof. The oil may be anyvegetable oil, fish oil, animal oil or synthetically prepared oil thatcan be metabolized by the body of the subject to which the emulsion willbe administered, and is not toxic to the subject. The subject may be ananimal, typically a mammal, and preferably a human.

In certain embodiments, the oil core is in liquid phase at 25° C. Theoil core is in liquid phase at 25° C., when it displays the propertiesof a fluid (as distinguished from solid and gas; and having a definitevolume but no definite shape) when stored at 25° C. The emulsion,however, may be stored and used at any suitable temperature. Preferably,the oil core is in liquid phase at 4° C.

The oil may be any long chain alkane, alkene or alkyne, or an acid oralcohol derivative thereof either as the free acid, its salt or an estersuch as a mono-, or di- or triester, such as the triglycerides andesters of 1,2-propanediol or similar poly-hydroxy alcohols. Alcohols maybe acylated employing a mono- or poly-functional acid, for exampleacetic acid, propanoic acid, citric acid or the like. Ethers derivedfrom long chain alcohols which are oils and meet the other criteria setforth herein may also be used.

The individual alkane, alkene or alkyne moiety and its acid or alcoholderivatives will generally have from about 6 to about 30 carbon atoms.The moiety may have a straight or branched chain structure. It may befully saturated or have one or more double or triple bonds. Where monoor poly ester- or ether-based oils are employed, the limitation of about6 to about 30 carbons applies to the individual fatty acid or fattyalcohol moieties, not the total carbon count.

It is particularly desirable that the oil can be metabolized by the hostto which the emulsion is administered.

Any suitable oils from an animal, fish or vegetable source may be used.Sources for vegetable oils include nuts, seeds and grains, and suitableoils, such as, peanut oil, soybean oil, coconut oil, and olive oil andthe like. Other suitable seed oils include safflower oil, cottonseedoil, sunflower seed oil, sesame seed oil and the like. In the graingroup, corn oil, and the oil of other cereal grains such as wheat, oats,rye, rice, teff, triticale and the like may also be used. The technologyfor obtaining vegetable oils is well developed and well known. Thecompositions of these and other similar oils may be found in, forexample, the Merck Index, and source materials on foods, nutrition andfood technology.

About six to about ten carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. These products arecommercially available under the name NEOBEES from PVO International,Inc., Chemical Specialties Division, 416 Division Street, Boongon, N.J.and others.

Animal oils and fats are often in solid phase at physiologicaltemperatures due to the fact that they exist as triglycerides and have ahigher degree of saturation than oils from fish or vegetables. However,fatty acids are obtainable from animal fats by partial or completetriglyceride saponification which provides the free fatty acids. Fatsand oils from mammalian milk are metabolizable and may therefore be usedin the practice of this invention. The procedures for separation,purification, saponification and other means necessary for obtainingpure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene),a branched, unsaturated terpenoid, is particularly preferred herein. Amajor source of squalene is shark liver oil, although plant oils(primarily vegetable oils), including amaranth seed, rice bran, wheatgerm, and olive oils, are also suitable sources. Squalene can also beobtained from yeast or other suitable microbes. In some embodiments,Squalene is preferably obtained from non-animal sources, such as fromolives, olive oil or yeast. Squalane, the saturated analog to squalene,is also preferred. Fish oils, including squalene and squalane, arereadily available from commercial sources or may be obtained by methodsknown in the art.

In certain embodiments, the oil core comprises an oil that is selectedfrom the group consisting of: Castor oil, Coconut oil, Corn oil,Cottonseed oil, Evening primrose oil, Fish oil, Jojoba oil, Lard oil,Linseed oil, Olive oil, Peanut oil, Safflower oil, Sesame oil, Soybeanoil, Squalene, Squalane, Sunflower oil, Wheatgerm oil, and Mineral oil.In exemplary embodiments, the oil core comprises Soybean oil, Sunfloweroil, Olive oil, Squalene, Squalane or a combination thereof. Squalanecan also be used as the oil. In exemplary embodiments, the oil corecomprises Squalene, Squalane, or a combination thereof.

The oil component of the emulsion may be present in an amount from about0.2% to about 20% (v/v). For example, the cationic oil-in-water emulsionmay comprise from about 0.2% to about 20% (v/v) oil, from about 0.2% toabout 15% (v/v) oil, from about 0.2% to about 10% (v/v) oil, from about0.2% to about 9% (v/v) oil, from about 0.2% to about 8% (v/v) oil, fromabout 0.2% to about 7% (v/v) oil, from about 0.2% to about 6% (v/v) oil,from about 0.2% to about 5% (v/v) oil, from about 0.2% to about 4.3%(v/v) oil, from about 0.3% to about 20% (v/v) oil, from about 0.4% toabout 20% (v/v) oil, from about 0.5% to about 20% (v/v) oil, from about1% to about 20% (v/v) oil, from about 2% to about 20% (v/v) oil, fromabout 3% to about 20% (v/v) oil, from about 4% to about 20% (v/v) oil,from about 4.3% to about 20% (v/v) oil, from about 5% to about 20% (v/v)oil, about 0.5% (v/v) oil, about 1% (v/v) oil, about 1.5% (v/v) oil,about 2% (v/v) oil, about 2.5% (v/v) oil, about 3% (v/v) oil, about 3.5%(v/v) oil, about 4% (v/v) oil, about 4.3% (v/v) oil, about 5% (v/v) oil,or about 10% (v/v) oil.

Alternatively, the cationic oil-in-water emulsion may comprise fromabout 0.2% to about 10% (w/v) oil, from about 0.2% to about 9% (w/v)oil, from about 0.2% to about 8% (w/v) oil, from about 0.2% to about 7%(w/v) oil, from about 0.2% to about 6% (w/v) oil, from about 0.2% toabout 5% (w/v) oil, from about 0.2% to about 4.3% (w/v) oil, or about4.3% (w/v) oil.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 0.5% (v/v) oil. In another exemplary embodiment, the cationicoil-in-water emulsion comprises about 4.3% (v/v) oil. In anotherexemplary embodiment, the cationic oil-in-water emulsion comprises about5% (v/v) oil. In another exemplary embodiment, the cationic oil-in-wateremulsion comprises about 4.3% (w/v) squalene.

As noted above, the percentage of oil described above is determinedbased on the initial amount of the oil that is used to prepare theemulsions. It is understood in the art that the actual concentration ofthe oil in the final product (e.g., a packaged, sterilized emulsion thatis ready for administration) might be slightly lower, sometimes up toabout 10% or about 20%.

B. Cationic Lipids

The emulsion particles described herein comprise a cationic lipid, whichcan interact with the negatively charged molecule thereby anchoring themolecule to the emulsion particles.

The cationic lipid can have a positive charge at about 12 pH, about 11pH, about 10 pH, about 9 pH, about 8 pH, about 7 pH, or about 6 pH.

Any suitable cationic lipid may be used. Generally, the cationic lipidcontains a nitrogen atom that is positively charged under physiologicalconditions. Suitable cationic lipids include, benzalkonium chloride(BAK), benzethonium chloride, cetrimide (which containstetradecyltrimethylammonium bromide and possibly small amounts ofdodecyltrimethylammonium bromide and hexadecyltrimethyl ammoniumbromide), cetylpyridinium chloride (CPC), cetyl trimethylammoniumchloride (CTAC), primary amines, secondary amines, tertiary amines,including but not limited to N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, other quaternary amine salts,including but not limited to dodecyltrimethylammonium bromide,hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammoniumbromide, benzyldimethyldodecylammonium chloride,benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethoniumchloride, methyl mixed trialkyl ammonium chloride, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemetha-naminiumchloride (DEBDA), dialkyldimetylammonium salts,[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio) propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl,distearoyl, dioleoyl),1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide andcetylpyridinium chloride), N-alkylpiperidinium salts, dicationicbolaform electrolytes (C₁₂Me₆; C₁₂Bu₆),dialkylglycetylphosphorylcholine, lysolecithin, L-αdioleoylphosphatidylethanolamine, cholesterol hemisuccinate cholineester, lipopolyamines, including but not limited todioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine(LPLL, LPDL), poly (L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives ofcholesterol, including but not limited to cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamidoethylenedimethylamine, cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, cholesteryl-3β-carboxyamidoethylenedimethylamine, and3γ-[N—(N′,N-dimethylaminoetanecarbomoyl]cholesterol) (DC-Cholesterol),1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), and combination thereof.

Other cationic lipids suitable for use in the invention include, e.g.,the cationic lipids described in U.S. Patent Publications 2008/0085870(published Apr. 10, 2008) and 2008/0057080 (published Mar. 6, 2008).

Other cationic lipids suitable for use in the invention include, e.g.,Lipids E0001-E0118 or E0119-E0180 as disclosed in Table 6 (pages112-139) of WO 2011/076807 (which also discloses methods of making, andmethod of using these cationic lipids). Additional suitable cationiclipids include N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).

The emulsion may comprise any combination of two or more of the cationiclipids described herein.

In some embodiments, the cationic lipid contains a quaternary amine.

In preferred embodiments, the cationic lipid is selected from the groupconsisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), Lipids E0001-E0118 orE0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011/076807,and combinations thereof.

In other preferred embodiments, the cationic lipid is selected from thegroup consisting of 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), Lipids E0001-E0118or E0119-E0180 as disclosed in Table 6 (pages 112-139) of WO 2011/076807(incorporated herein by reference), and combinations thereof.

In certain embodiments, the cationic lipid is DOTAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOTAP. For example, the cationic oil-in-water emulsion maycomprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, from about 0.8mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5mg/ml, about 1.6 mg/ml, about 12 mg/ml, about 18 mg/ml, about 20 mg/ml,about 21.8 mg/ml, about 24 mg/ml, etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DC Cholesterol. Thecationic oil-in-water emulsion may comprise DC Cholesterol at from about0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationicoil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/mlto about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, fromabout 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml toabout 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, fromabout 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml toabout 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, fromabout 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml toabout 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml,about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml,about 4.92 mg/ml, etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.62 mg/ml to about 4.92 mg/ml DC Cholesterol, such as 2.46mg/ml.

In certain embodiments, the cationic lipid is DDA. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/mlDDA. For example, the cationic oil-in-water emulsion may comprise DDA atfrom about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.5mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml toabout 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1mg/ml to about 2.5 mg/ml, from about 0.1 mg/ml to about 2 mg/ml, fromabout 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml toabout 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, fromabout 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml toabout 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, fromabout 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml,from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45mg/ml, etc. Alternatively, the cationic oil-in-water emulsion maycomprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about21.6 mg/ml, about 25 mg/ml.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.73 mg/ml to about 1.45 mg/ml DDA, such as 1.45 mg/ml.

In certain embodiments, the cationic lipid is DOTMA. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOTMA. For example, the cationic oil-in-water emulsion maycomprise DOTMA at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, fromabout 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml toabout 1.6 mg/ml, from about 0.8 mg/ml to about 1.6 mg/ml, from about 0.8mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.35 mg/ml, about 1.4mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12 mg/ml, about 18 mg/ml,about 20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DOTMA, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DOEPC. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 25mg/ml DOEPC. For example, the cationic oil-in-water emulsion maycomprise DOEPC at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.8 mg/ml DOEPC, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml, 1.6 mg/ml, 1.7 mg/ml, or 1.8 mg/ml.

In certain embodiments, the cationic lipid is DSTAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DSTAP. For example, the cationic oil-in-water emulsion maycomprise DSTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DSTAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In certain embodiments, the cationic lipid is DODAC. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DODAC. For example, the cationic oil-in-water emulsion maycomprise DODAC at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.15 mg/ml, about 1.16 mg/ml, about 1.17 mg/ml,about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml,about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml,about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about 20 mg/ml, about22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom 0.73 mg/ml to about 1.45 mg/ml DODAC, such as 1.45 mg/ml.

In certain embodiments, the cationic lipid is DODAP. The cationicoil-in-water emulsion may comprise from about 0.5 mg/ml to about 50mg/ml DODAP. For example, the cationic oil-in-water emulsion maycomprise DODAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, fromabout 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml toabout 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, fromabout 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml toabout 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fromabout 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml toabout 5 mg/ml, from about 0.5 mg/ml to about 4 mg/ml, from about 0.5mg/ml to about 3 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, fromabout 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml toabout 1.6 mg/ml, from about 0.6 mg/ml to about 1.7 mg/ml, from about 0.7mg/ml to about 1.7 mg/ml, from about 0.8 mg/ml to about 1.7 mg/ml, fromabout 0.8 mg/ml to about 3.0 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml,about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml,about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml,about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml,about 1.9 mg/ml, about 2.0 mg/ml, about 12 mg/ml, about 18 mg/ml, about20 mg/ml, about 22.5 mg/ml, about 25 mg/ml etc.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesfrom about 0.8 mg/ml to about 1.6 mg/ml DODAP, such as 0.8 mg/ml, 1.2mg/ml, 1.4 mg/ml or 1.6 mg/ml.

In some cases, it may be desirable to use a cationic lipid that issoluble in the oil core. For example, DOTAP, DOEPC, DODAC, and DOTMA aresoluble in squalene or squalane. In other cases, it may be desirable touse a cationic lipid that is not soluble in the oil core. For example,DDA and DSTAP are not soluble in squalene. It is within the knowledge inthe art to determine whether a particular lipid is soluble or insolublein the oil and choose an appropriate oil and lipid combinationaccordingly. For example, solubility can be predicted based on thestructures of the lipid and oil (e.g., the solubility of a lipid may bedetermined by the structure of its tail). For example, lipids having oneor two unsaturated fatty acid chains (e.g., oleoyl tails), such asDOTAP, DOEPC, DODAC, DOTMA, are soluble in squalene or squalane; whereaslipids having saturated fatty acid chains (e.g., stearoyl tails) are notsoluble in squalene. Alternatively, solubility can be determinedaccording to the quantity of the lipid that dissolves in a givenquantity of the oil to form a saturated solution.

As noted above, the concentration of a lipid described above isdetermined based on the initial amount of the lipid that is used toprepare the emulsions. It is understood in the art that the actualconcentration of the oil in the final product (e.g., a packaged,sterilized emulsion that is ready for administration) might be slightlylower, sometimes by up to about 20%.

C. Additional Components

The cationic oil-in-water emulsions described herein may furthercomprise additional components. For example, the emulsions may comprisecomponents that can promote particle formation, improve the complexationbetween the nucleic acid molecules and the cationic particles, orincrease the stability of the nucleic acid molecule (e.g., to preventdegradation of an RNA molecule). If desired, the cationic oil-in-wateremulsion can contain an antioxidant, such as citrate, ascorbate or saltsthereof.

Surfactants

In certain embodiments, the particles of the cationic oil-in-wateremulsion further comprise a surfactant.

A substantial number of surfactants have been used in the pharmaceuticalsciences. These include naturally derived materials such as gums fromtrees, vegetable protein, sugar-based polymers such as alginates andcellulose, and the like. Certain oxypolymers or polymers having ahydroxide or other hydrophilic substituent on the carbon backbone havesurfactant activity, for example, povidone, polyvinyl alcohol, andglycol ether-based mono- and poly-functional compounds. Long chainfatty-acid-derived compounds form a third substantial group ofemulsifying and suspending agents which could be used in this invention.

Specific examples of suitable surfactants include the following:

1. Water-soluble soaps, such as the sodium, potassium, ammonium andalkanol-ammonium salts of higher fatty acids (C₁₀-C₂₂), in particularsodium and potassium tallow and coconut soaps.

2. Anionic synthetic non-soap surfactants, which can be represented bythe water-soluble salts of organic sulfuric acid reaction productshaving in their molecular structure an alkyl radical containing fromabout 8 to 22 carbon atoms and a radical selected from the groupconsisting of sulfonic acid and sulfuric acid ester radicals. Examplesof these are the sodium or potassium alkyl sulfates, derived from tallowor coconut oil; sodium or potassium alkyl benzene sulfonates; sodiumalkyl glyceryl ether sulfonates; sodium coconut oil fatty acidmonoglyceride sulfonates and sulfates; sodium or potassium salts ofsulfuric acid esters of the reaction product of one mole of a higherfatty alcohol and about 1 to 6 moles of ethylene oxide; sodium orpotassium alkyl phenol ethylene oxide ether sulfonates, with 1 to 10units of ethylene oxide per molecule and in which the alkyl radicalscontain from 8 to 12 carbon atoms; the reaction product of fatty acidsesterified with isethionic acid and neutralized with sodium hydroxide;sodium or potassium salts of fatty acid amide of a methyl tauride; andsodium and potassium salts of SO₃-sulfonated C₁₀-C₂₄ α-olefins.

3. Nonionic synthetic surfactants made by the condensation of alkyleneoxide groups with an organic hydrophobic compound. Typical hydrophobicgroups include condensation products of propylene oxide with propyleneglycol, alkyl phenols, condensation product of propylene oxide andethylene diamine, aliphatic alcohols having 8 to 22 carbon atoms, andamides of fatty acids.

4. Nonionic surfactants, such as amine oxides, phosphine oxides andsulfoxides, having semipolar characteristics. Specific examples of longchain tertiary amine oxides include dimethyldodecylamine oxide andbis-(2-hydroxyethyl) dodecylamine. Specific examples of phosphine oxidesare found in U.S. Pat. No. 3,304,263, issued Feb. 14, 1967, and includedimethyldodecylphosphine oxide and dimethyl-(2hydroxydodecyl) phosphineoxide.

5. Long chain sulfoxides, including those corresponding to the formulaR¹—SO—R² wherein R¹ and R² are substituted or unsubstituted alkylradicals, the former containing from about 10 to about 28 carbon atoms,whereas R² contains from 1 to 3 carbon atoms. Specific examples of thesesulfoxides include dodecyl methyl sulfoxide and 3-hydroxy tridecylmethyl sulfoxide.

6. Ampholytic synthetic surfactants, such as sodium3-dodecylaminopropionate and sodium 3-dodecylaminopropane sulfonate.

7. Zwitterionic synthetic surfactants, such as3-(N,N-dimethyl-N-hexadecylammonio)propane-1-sulfonate and3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy propane-1-sulfonate.

Additionally, all of the following types of surfactants can be used in acomposition of the present invention: (a) soaps (i.e., alkali salts) offatty acids, rosin acids, and tall oil; (b) alkyl arene sulfonates; (c)alkyl sulfates, including surfactants with both branched-chain andstraight-chain hydrophobic groups, as well as primary and secondarysulfate groups; (d) sulfates and sulfonates containing an intermediatelinkage between the hydrophobic and hydrophilic groups, such as thefatty acylated methyl taurides and the sulfated fatty monoglycerides;(e) long-chain acid esters of polyethylene glycol, especially the talloil esters; (f) polyethylene glycol ethers of alkylphenols; (g)polyethylene glycol ethers of long-chain alcohols and mercaptans; and(h) fatty acyl diethanol amides. Since surfactants can be classified inmore than one manner, a number of classes of surfactants set forth inthis paragraph overlap with previously described surfactant classes.

There are a number of surfactants specifically designed for and commonlyused in biological situations. Such surfactants are divided into fourbasic types: anionic, cationic, zwitterionic (amphoteric), and nonionic.Exemplary anionic surfactants include, e.g., perfluorooctanoate (PFOA orPFO), perfluorooctanesulfonate (PFOS), alkyl sulfate salts such assodium dodecyl sulfate (SDS) or ammonium lauryl sulfate, sodium laurethsulfate (also known as sodium lauryl ether sulfate, SLES), alkyl benzenesulfonate, and fatty acid salts. Exemplary cationic surfactants include,e.g., alkyltrimethylammonium salts such as cetyl trimethylammoniumbromide (CTAB, or hexadecyl trimethyl ammonium bromide), cetylpyridiniumchloride (CPC), polyethoxylated tallow amine (POEA), benzalkoniumchloride (BAC), benzethonium chloride (BZT). Exemplary zwitterionic(amphoteric) surfactants include, e.g., dodecyl betaine, cocamidopropylbetaine, and coco ampho glycinate. Exemplary nonionic surfactantsinclude, e.g., alkyl poly(ethylene oxide), alkylphenol poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide)(commercially called poloxamers or poloxamines), Aayl polyglucosides(e.g., octyl glucoside or decyl maltoside), fatty alcohols (e.g., cetylalcohol or oleyl alcohol), cocamide MEA, cocamide DEA, Pluronic® F-68(polyoxyethylene-polyoxypropylene block copolymer), and polysorbates,such as Tween 20 (polysorbate 20), Tween 80 (polysorbate 80;polyoxyethylenesorbitan monooleate), dodecyl dimethylamine oxide, andvitamin E tocopherol propylene glycol succinate (Vitamin E TPGS).

A particularly useful group of surfactants are the sorbitan-basednon-ionic surfactants. These surfactants are prepared by dehydration ofsorbitol to give 1,4-sorbitan which is then reacted with one or moreequivalents of a fatty acid. The fatty-acid-substituted moiety may befurther reacted with ethylene oxide to give a second group ofsurfactants.

The fatty-acid-substituted sorbitan surfactants are made by reacting1,4-sorbitan with a fatty acid such as lauric acid, palmitic acid,stearic acid, oleic acid, or a similar long chain fatty acid to give the1,4-sorbitan mono-ester, 1,4-sorbitan sesquiester or 1,4-sorbitantriester. The common names for these surfactants include, for example,sorbitan monolaurate, sorbitan monopalmitate, sorbitan monoestearate,sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate.These surfactants are commercially available under the name SPAN® orARLACEL®, usually with a letter or number designation whichdistinguishes between the various mono, di- and triester substitutedsorbitans.

SPAN® and ARLACEL® surfactants are hydrophilic and are generally solubleor dispersible in oil. They are also soluble in most organic solvents.In water they are generally insoluble but dispersible. Generally thesesurfactants will have a hydrophilic-lipophilic balance (HLB) numberbetween 1.8 to 8.6. Such surfactants can be readily made by means knownin the art or are commercially available.

A related group of surfactants comprises olyoxyethylene sorbitanmonoesters and olyoxyethylene sorbitan triesters. These materials areprepared by addition of ethylene oxide to a 1,4-sorbitan monester ortriester. The addition of polyoxyethylene converts the lipophilicsorbitan mono- or triester surfactant to a hydrophilic surfactantgenerally soluble or dispersible in water and soluble to varying degreesin organic liquids.

These materials, commercially available under the mark TWEEN®, areuseful for preparing oil-in-water emulsions and dispersions, or for thesolubilization of oils and making anhydrous ointments water-soluble orwashable. The TWEEN® surfactants may be combined with a related sorbitanmonester or triester surfactants to promote emulsion stability. TWEEN®surfactants generally have a HLB value falling between 9.6 to 16.7.TWEEN® surfactants are commercially available.

A third group of non-ionic surfactants which could be used alone or inconjunction with SPAN®, ARLACEL® and TWEEN® surfactants are thepolyoxyethylene fatty acids made by the reaction of ethylene oxide witha long-chain fatty acid. The most commonly available surfactant of thistype is solid under the name MYRJ® and is a polyoxyethylene derivativeof stearic acid. MYRJ® surfactants are hydrophilic and soluble ordispersible in water like TWEEN® surfactants. The MYRJ® surfactants maybe blended with TWEEN® surfactants or with TWEEN®/SPAN® or ARLACEL®surfactant mixtures for use in forming emulsions. MYRJ® surfactants canbe made by methods known in the art or are available commercially.

A fourth group of polyoxyethylene based non-ionic surfactants are thepolyoxyethylene fatty acid ethers derived from lauryl, acetyl, stearyland oleyl alcohols. These materials are prepared as above by addition ofethylene oxide to a fatty alcohol. The commercial name for thesesurfactants is BRIJ®. BRIJ® surfactants may be hydrophilic or lipophilicdepending on the size of the polyoxyethylene moiety in the surfactant.While the preparation of these compounds is available from the art, theyare also readily available from commercial sources.

Other non-ionic surfactants which could potentially be used are, forexample, polyoxyethylene, polyol fatty acid esters, polyoxyethyleneether, polyoxypropylene fatty ethers, bee's wax derivatives containingpolyoxyethylene, polyoxyethylene lanolin derivative, polyoxyethylenefatty glycerides, glycerol fatty acid esters or other polyoxyethyleneacid alcohol or ether derivatives of long-chain fatty acids of 12-22carbon atoms.

As the emulsions and formulations of the invention are intended to bemulti-phase systems, it is preferable to choose an emulsion-formingnon-ionic surfactant which has an HLB value in the range of about 7 to16. This value may be obtained through the use of a single non-ionicsurfactant such as a TWEEN® surfactant or may be achieved by the use ofa blend of surfactants such as with a sorbitan mono, di- or triesterbased surfactant; a sorbitan ester polyoxyethylene fatty acid; asorbitan ester in combination with a polyoxyethylene lanolin derivedsurfactant; a sorbitan ester surfactant in combination with a high HLBpolyoxyethylene fatty ether surfactant; or a polyethylene fatty ethersurfactant or polyoxyethylene sorbitan fatty acid.

In certain embodiments, the emulsion comprises a single non-ionicsurfactant, most particularly a TWEEN® surfactant, as the emulsionstabilizing non-ionic surfactant. In an exemplary embodiment, theemulsion comprises TWEEN® 80, otherwise known as polysorbate 80 orpolyoxyethylene 20 sorbitan monooleate. In other embodiments, theemulsion comprises two or more non-ionic surfactants, in particular aTWEEN® surfactant and a SPAN® surfactant. In an exemplary embodiment,the emulsion comprises TWEEN® 80 and SPAN®85.

The oil-in-water emulsions can contain from about 0.01% to about 2.5%surfactant (v/v or w/v), about 0.01% to about 2% surfactant, 0.01% toabout 1.5% surfactant, 0.01% to about 1% surfactant, 0.01% to about 0.5%surfactant, 0.05% to about 0.5% surfactant, 0.08% to about 0.5%surfactant, about 0.08% surfactant, about 0.1% surfactant, about 0.2%surfactant, about 0.3% surfactant, about 0.4% surfactant, about 0.5%surfactant, about 0.6% surfactant, about 0.7% surfactant, about 0.8%surfactant, about 0.9% surfactant, or about 1% surfactant.

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% Tween 80 (polysorbate 80;polyoxyethylenesorbitan monooleate).

In an exemplary embodiment, the oil-in-water emulsion contains 0.08%Tween 80.

Alternatively or in addition, the oil-in-water emulsions can contain0.05% to about 1%, 0.05% to about 0.9%, 0.05% to about 0.8%, 0.05% toabout 0.7%, 0.05% to about 0.6%, 0.05% to about 0.5%, about 0.08%, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% SPAN85 (sorbtian trioleate).

Alternatively or in addition, the oil-in-water emulsions can contain acombination of surfactants described herein. For example, a combinationof Tween 80 (polysorbate 80; polyoxyethylenesorbitan monooleate) andSPAN85 (sorbtian trioleate) may be used. The emulsions may containvarious amounts Tween® 80 and SPAN85 (e.g., those exemplified above),including equal amounts of these surfactants. For example, theoil-in-water emulsions can contain about 0.05% Tween® 80 and about 0.05%SPAN®85, about 0.1% Tween® 80 and about 0.1% SPAN®85, about 0.2% Tween®80 and about 0.2% SPAN®85, about 0.3% Tween® 80 and about 0.3% SPAN®85,about 0.4% Tween® 80 and about 0.4% SPAN®85, about 0.5% Tween® 80 andabout 0.5% SPAN®85, about 0.6% Tween® 80 and about 0.6% SPAN®85, about0.7% Tween® 80 and about 0.7% SPAN®85, about 0.8% Tween® 80 and about0.8% SPAN®85, about 0.9% Tween® 80 and about 0.9% SPAN®85, or about 1%Tween® 80 and about 1.0% SPAN®85.

Polyethylene Glycol (PEG)-lipids, such as PEG coupled todialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG),PEG coupled to phosphatidylethanolamine (PE) (PEG-PE) or some otherphospholipids (PEG-phospholipids), PEG conjugated to ceramides(PEG-Cer), or a combination thereof, may also be used as surfactants(see, e.g., U.S. Pat. No. 5,885,613; U.S. patent application publicationNos. 2003/0077829, 2005/0175682 and 2006/0025366). Other suitablePEG-lipids include, e.g., PEG-dialkyloxypropyl (DAA) lipids orPEG-diacylglycerol (DAG) lipids. Exemplary PEG-DAG lipids include, e.g.,PEG-dilauroylglycerol (C₁₂) lipids, PEG-dimyristoylglycerol (C₁₄)lipids, PEG-dipalmitoylglycerol (C₁₆) lipids, or PEG-distearoylglycerol(C₁₈) lipids. Exemplary PEG-DAA lipids include, e.g.,PEG-dilauryloxypropyl (C₁₂) lipids, PEG-dimyristyloxypropyl (C₁₄)lipids, PEG-dipalmityloxypropyl (C₁₆) lipids, or PEG-distearyloxypropyl(C₁₈) lipids.

PEGs are classified by their molecular weights; for example, PEG 2000has an average molecular weight of about 2,000 daltons, and PEG 5000 hasan average molecular weight of about 5,000 daltons. PEGs arecommercially available from Sigma Chemical Co. as well as othercompanies and include, for example, the following:monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Inaddition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH₂COOH), isparticularly useful for preparing the PEG-lipid conjugates including,e.g., PEG-DAA conjugates.

Preferably, the PEG has an average molecular weight of from about 1000to about 5000 daltons (e.g., PEG₁₀₀₀, PEG₂₀₀₀, PEG₃₀₀₀, PEG₄₀₀₀,PEG₅₀₀₀). The PEG can be optionally substituted by an alkyl, alkoxy,acyl or aryl group. PEG can be conjugated directly to the lipid or maybe linked to the lipid via a linker moiety. Any linker moiety suitablefor coupling the PEG to a lipid can be used including, e.g., non-estercontaining linker moieties and ester-containing linker moieties

In exemplary embodiments, PEG₂₀₀₀PE, PEG₅₀₀₀PE, PEG₁₀₀₀DMG, PEG₂₀₀₀DMG,PEG₃₀₀₀DMG, or a combination thereof, is used as a surfactant. Incertain exemplary embodiments, the oil-in-water emulsion contains fromabout 1 mg/ml to about 80 mg/ml PEG₂₀₀₀PE, PEG₅₀₀₀PE, PEG₁₀₀₀DMG,PEG₂₀₀₀DMG, or PEG₃₀₀₀DMG.

Phospholipids

In certain embodiments, the particles of the cationic oil-in-wateremulsion further comprise a phospholipid.

Phospholipids are esters of fatty acids in which the alcohol componentof the molecule contains a phosphate group. Phospholipids includeglycerophosphatides (containing glycerol) and the sphingomyelins(containing sphingosine). Exemplary phospholipids includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine andsphingomyelin; and synthetic phospholipids comprising dimyristoylphosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoylphosphatidylcholine, distearoyl phosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoyl phosphatidylserine, distearoylphosphatidylserine, and dipalmitoyl serine.

The following exemplary phopholipids may be used.

DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA-NA1,2-Dierucoyl-sn-Glycero-3-Phosphate(Sodium Salt) DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG-NA1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA-NA1,2-Dilauroyl-sn-Glycero-3-Phosphate(Sodium Salt) DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG-NA1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) (SodiumSalt) DLPG-NH4 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol.. .) DLPS-NA 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DMPA-NA 1,2-Diimyristoyl-sn-Glycero-3-Phosphate(Sodium Salt) DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG-NA1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DMPG-NH41,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .)DMPG-NH4/NA 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. ..) DMPS-NA 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DOPA-NA 1,2-Dioleoyl-sn-Glycero-3-Phosphate(Sodium Salt) DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DOPS-NA1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) DPPA-NA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate(Sodium Salt) DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG-NA1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DPPG-NH41,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DPPS-NA1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) DPyPE1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA-NA1,2-Distearoyl-sn-Glycero-3-Phosphate(Sodium Salt) DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG-NA1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DSPG-NH41,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol. . .) DSPS-NA1,2-Distearoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) EPC Egg-PCHEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPCHydrogenated Soy PC LYSOPC MYRISTIC1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MPPC MSPC1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol). . .](SodiumSalt) PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

In certain embodiments, it may be advantageous to use a neutral lipid.It may also be advantageous to use a phospholipid, including azwitterionic phospholipid, for example, a phospholipid containing one ormore alkyl or alkenyl radicals of about 12 to about 22 carbons in length(e.g., about 12 to about 14, to about 16, to about 18, to about 20, toabout 22 carbons), which radicals may contain, for example, from 0 to 1to 2 to 3 double bonds. It may be advantageous to use a zwitterionicphospholipid.

Preferred phospholipids include, e.g.,1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), Eggphosphatidylcholine (egg PC), palmitoyl oleoyl phosphatidylcholine(POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), DPPC, dipalmitoyl phosphatidylcholine(DPPC), palmitoyl linoleyl phosphatidylcholine (PLPC), DPyPE, or acombination thereof.

In certain embodiments, the phospholipid is DOPE. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml DOPE. For example, the cationic oil-in-water emulsion may compriseDOPE at from about 0.5 mg/ml to about 10 mg/ml, from about 0.1 mg/ml toabout 10 mg/ml, or from about 1.5 mg/ml to about 7.5 mg/ml DOPE.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.5 mg/ml DOPE.

In certain embodiments, the phospholipid is egg PC. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml egg PC. For example, the cationic oil-in-water emulsion maycomprise egg PC at from about 0.1 mg/ml to about 10 mg/ml, from about1.0 mg/ml to about 10 mg/ml, or from about 1.5 mg/ml to about 3.5 mg/mlegg PC.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.55 mg/ml egg PC.

In certain embodiments, the phospholipid is DPyPE. The cationicoil-in-water emulsion may comprise from about 0.1 mg/ml to about 20mg/ml DPyPE. For example, the cationic oil-in-water emulsion maycomprise DPyPE at from about 0.1 mg/ml to about 10 mg/ml, from about 1.5mg/ml to about 10 mg/ml, or from about 1.5 mg/ml to about 5 mg/ml DPyPE.

In an exemplary embodiment, the cationic oil-in-water emulsion comprisesabout 1.6 mg/ml DPyPE.

In certain embodiments, the emulsion particles may comprise acombination of a surfactant and a phospholipid described herein.

D. Aqueous Phase (Continuous Phase)

The aqueous phase (continuous phase) of the oil-in-water emulsions is abuffered salt solution (e.g., saline) or water. The buffered saltsolution is an aqueous solution that comprises a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), and can further comprise an osmolalityadjusting agent (e.g., a saccharide), a polymer, a surfactant, or acombination thereof. The aqueous phase can contain an antioxidant, suchas citrate, ascorbate or salts thereof. If the emulsions are formulatedfor parenteral administration, it is preferable to make up finalbuffered solutions so that the tonicity, i.e., osmolality, isessentially the same as normal physiological fluids in order to preventundesired post-administration consequences, such as post-administrationswelling or rapid absorption of the composition. It is also preferableto buffer the aqueous phase in order to maintain a pH compatible withnormal physiological conditions. Also, in certain instances, it may bedesirable to maintain the pH at a particular level in order to insurethe stability of certain components of the emulsion.

For example, it may be desirable to prepare an emulsion that is isotonic(i.e., the same permeable solute (e.g., salt) concentration as thenormal cells of the body and the blood) and isosmotic. To controltonicity, the emulsion may comprise a physiological salt, such as asodium salt. Sodium chloride (NaCl), for example, may be used at about0.9% (w/v) (physiological saline). Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate, magnesium chloride, calcium chloride, etc. Non-ionictonicifying agents can also be used to control tonicity. A number ofnon-ionic tonicity modifying agents ordinarily known to those in theart. These are typically carbohydrates of various classifications (see,for example, Voet and Voet (1990) Biochemistry (John Wiley & Sons, NewYork). Monosaccharides classified as aldoses such as glucose, mannose,arabinose, and ribose, as well as those classified as ketoses such asfructose, sorbose, and xylulose can be used as non-ionic tonicifyingagents in the present invention. Disaccharides such a sucrose, maltose,trehalose, and lactose can also be used. In addition, alditols (acyclicpolyhydroxy alcohols, also referred to as sugar alcohols) such asglycerol, mannitol, xylitol, and sorbitol are non-ionic tonicifyingagents useful in the present invention. Non-ionic tonicity modifyingagents can be present at a concentration of from about 0.1% to about 10%or about 1% to about 10%, depending upon the agent that is used.

The aqueous phase may be buffered. Any physiologically acceptable buffermay be used herein, such as water, citrate buffers, phosphate buffers,acetate buffers, tris buffers, bicarbonate buffers, carbonate buffers,succinate buffer, or the like. The pH of the aqueous component willpreferably be between 6.0-8.0, preferably about 6.2 to about 6.8. In anexemplary embodiment, the buffer is 10 mM citrate buffer with a pH at6.5. In another exemplary embodiment, the aqueous phase is, or thebuffer prepared using, RNase-free water or DEPC treated water. In somecases, high salt in the buffer might interfere with complexation ofnucleic acid molecule to the emulsion particle therefore is avoided. Inother cases, certain amount of salt in the buffer may be included.

In an exemplary embodiment, the buffer is 10 mM citrate buffer with a pHat 6.5. In another exemplary embodiment, the aqueous phase is, or thebuffer is prepared using, RNase-free water or DEPC treated water.

The aqueous phase may also comprise additional components such asmolecules that change the osmolarity of the aqueous phase or moleculesthat stabilizes the nucleic acid molecule after complexation.Preferably, the osmolarity of the aqueous phase is adjusting using anon-ionic tonicifying agent, such as a sugar (e.g., trehalose, sucrose,dextrose, fructose, reduced palatinose, etc.), a sugar alcohol (such asmannitol, sorbitol, xylitol, erythritol, lactitol, maltitol, glycerol,etc.), or combinations thereof. If desired, a nonionic polymer (e.g., apoly(alkyl glycol) such as polyethylene glycol, polypropylene glycol, orpolybutlyene glycol) or nonionic surfactant can be used.

In some case, unadulterated water may be preferred as the aqueous phaseof the emulsion when the emulsion is initially prepared. For example,increasing the salt concentration or sugar concentration may make itmore difficult to achieve the desirable particle size (e.g., less thanabout 200 nm).

In certain embodiments, the aqueous phase of the cationic oil-in-wateremulsion may further comprise a polymer or a surfactant, or acombination thereof. In an exemplary embodiment, the oil-in-wateremulsion contains a poloxamer. Poloxamers are nonionic triblockcopolymers having a central hydrophobic chain of polyoxypropylene(poly(propylene oxide)) flanked by two hydrophilic chains ofpolyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by thetrade name Pluronic® polymers. Poloxamer polymers may lead to greaterstability and increased RNase resistance of the RNA molecule after RNAcomplexation.

Alternatively or in addition, the cationic oil-in-water emulsion maycomprise from about 0.1% to about 20% (w/v) polymer, or from about 0.05%to about 10% (w/v) polymer. For example, the cationic oil-in-wateremulsion may comprise a polymer (e.g., a poloxamer such as Pluronic®F127) at from about 0.1% to about 20% (w/v), from about 0.1% to about10% (w/v), from about 0.05% to about 10% (w/v), or from about 0.05% toabout 5% (w/v).

In an exemplary embodiment, the oil-in-water emulsion comprises about 4%(w/v), or about 8% (w/v) Pluronic® F127.

The quantity of the aqueous component employed in these compositionswill be that amount necessary to bring the value of the composition tounity. That is, a quantity of aqueous component sufficient to make 100%will be mixed, with the other components listed above in order to bringthe compositions to volume.

4. Nucleic Acid Molecules

Although not wishing to be bound by any particular theory, it isbelieved that the nucleic acid molecules interact with the cationiclipid through non-covalent, ionic charge interactions (electrostaticforces), and the strength of the complex as well as the amount ofnucleic acid molecule that can be complexed to a particle are related tothe amount of cationic lipid in the particle. Additionally,hydrophobic/hydrophilic interactions between the nucleic acid moleculeand the surface of the particles may also play a role.

Nucleic acid molecules that can be complexed to the emulsion particlesinclude single or double stranded RNA or DNA. In preferred aspects, thenucleic acid molecule is an RNA molecule, such as an RNA that encodes apeptide, polypeptide or protein, including self-replicating RNAmolecules, or a small interfering RNA.

The complex can be formed by using techniques known in the art, examplesof which are described herein. For example, a nucleic acid-particlecomplex can be formed by mixing a cationic emulsion with the nucleicacid molecule, for example by vortexing. The amount of the nucleic acidmolecule and cationic lipid in the emulsions may be adjusted oroptimized to provide desired strength of binding and binding capacity.

For example, as described and exemplified herein, exemplary RNA-particlecomplexes were produced by varying the RNA:cationic lipid ratios (asmeasured by the “N/P ratio”). The term N/P ratio refers to the amount(moles) of protonatable nitrogen atoms in the cationic lipid divided bythe amount (moles) of phosphates on the RNA. The N/P ratio is at least,for example from 4:1 to 20:1 or from 4:1 to 15:1.

In some embodiments, the N/P ratio is from 1.1:1 to 20:1, 1.1:1 to 15:1,1.5:1 to 20:1, 1.5:1 to 15:1, 2:1 to 20:1, 2:1 to 15:1, 2.5:1 to 20:1,2.5:1 to 15:1, 3:1 to 20:1, 3:1 to 15:1, 3.5:1 to 20:1, or 3.5:1 to15:1.

The cationic oil-in-water emulsions described herein are particularlysuitable for formulating nucleic acid-based vaccines (e.g., DNAvaccines, RNA vaccines). The formation of a nucleic acid-emulsionparticle complex facilitates the uptake of the nucleic acid into hostcells, and protects the nucleic acid molecule from nuclease degradation.Transfected cells can then express the antigen encoded by the nucleicacid molecule, which can produce an immune response to the antigen. Likelive or attenuated viruses, nucleic acid-based vaccines can effectivelyengage both MHC-I and MHC-II pathways allowing for the induction of CD8⁺and CD4⁺ T cell responses, whereas antigen present in soluble form, suchas recombinant protein, generally induces only antibody responses.

The sequence of the nucleic acid molecule (e.g., RNA molecule) may becodon optimized or deoptimized for expression in a desired host, such asa human cell.

In certain embodiments, the nucleic acid molecule is an RNA molecule. Incertain embodiments, the RNA molecule encodes an antigen (peptide,polypeptide or protein) and the cationic oil in water emulsion issuitable for use as an RNA-based vaccine. The composition can containmore than one RNA molecule encoding an antigen, e.g., two, three, five,or ten RNA molecules that are complexed to the emulsion particles. Thatis, the composition can contain one or more different species of RNAmolecules, each encoding a different antigen. Alternatively or inaddition, one RNA molecule may also encode more than one antigen, e.g.,a bicistronic, or tricistronic RNA molecule that encodes different oridentical antigens. Accordingly, the cationic oil in water emulsion issuitable for use as an RNA-based vaccine, that is monovalent ormultivalent.

The sequence of the RNA molecule may be modified if desired, for exampleto increase the efficacy of expression or replication of the RNA, or toprovide additional stability or resistance to degradation. For example,the RNA sequence can be modified with respect to its codon usage, forexample, to increase translation efficacy and half-life of the RNA. Apoly A tail (e.g., of about 30 adenosine residues or more (SEQ ID NO:3)) may be attached to the 3′ end of the RNA to increase its half-life.The 5′ end of the RNA may be capped with a modified ribonucleotide withthe structure m7G (5′) ppp (5′) N (cap 0 structure) or a derivativethereof, which can be incorporated during RNA synthesis or can beenzymatically engineered after RNA transcription (e.g., by usingVaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase,guanylyl-transferase and guanine-7-methytransferase, which catalyzes theconstruction of N7-monomethylated cap 0 structures). Cap 0 structureplays an important role in maintaining the stability and translationalefficacy of the RNA molecule. The 5′ cap of the RNA molecule may befurther modified by a 2′-O-Methyltransferase which results in thegeneration of a cap 1 structure (m7Gppp [m2′-O]N), which may furtherincreases translation efficacy.

If desired, the RNA molecule can comprise one or more modifiednucleotides, in addition to any 5′ cap structure. There are more than 96naturally occurring nucleoside modifications found on mammalian RNA.See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196(1994). The preparation of nucleotides and modified nucleotides andnucleosides are well-known in the art, e.g. from U.S. Pat. Nos.4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524,5,132,418, 5,153,319, 5,262,530, 5,700,642 all of which are incorporatedby reference in their entirety herein, and many modified nucleosides andmodified nucleotides are commercially available.

Modified nucleobases which can be incorporated into modified nucleosidesand nucleotides and be present in the RNA molecules include: m5C(5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U(2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A(2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine);ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m1I (1-methylinosine);m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm(2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine);f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm(N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine);m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm(2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm(N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q(queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi(7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine);m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U(5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U(5-carboxymethylaminomethyluridine); cnmm5Um(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U(5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G(N2,N2,7-trimethylguano sine); m3Um (3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); m1Gm(1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine)irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14(4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine),hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof,dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-methyluracil, 5-(C₂-C₆)-alkenyluracil,5-(C₂-C₆)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil,5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,5-(C₁-C₆)-alkylcytosine, 5-methylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 7-deazaguanine, 8-azaguanine,7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine,7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine,2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine,7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen(abasic residue), m5C, m5U, m6A, s2U, W, or 2′-O-methyl-U. Many of thesemodified nucleobases and their corresponding ribonucleosides areavailable from commercial suppliers. See, e.g., WO 2011/005799 which isincorporated herein by reference.

A RNA used with the invention ideally includes only phosphodiesterlinkages between nucleosides, but in some embodiments it can containphosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

In some embodiments, the RNA molecule does not include modifiednucleotides, e.g., does not include modified nucleobases, and all of thenucleotides in the RNA molecule are conventional standardribonucleotides A, U, G and C, with the exception of an optional 5′ capthat may include, for example, 7-methylguanosine. In other embodiments,the RNA may include a 5′ cap comprising a 7′-methylguanosine, and thefirst 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ positionof the ribose.

A. Self-replicating RNA

In some aspects, the cationic oil in water emulsion contains aself-replicating RNA molecule. In certain embodiments, theself-replicating RNA molecule is derived from or based on an alphavirus.

Self-replicating RNA molecules are well known in the art and can beproduced by using replication elements derived from, e.g., alphaviruses,and substituting the structural viral proteins with a nucleotidesequence encoding a protein of interest. A self-replicating RNA moleculeis typically a (+)-strand molecule which can be directly translatedafter delivery to a cell, and this translation provides a RNA-dependentRNA polymerase which then produces both antisense and sense transcriptsfrom the delivered RNA. Thus the delivered RNA leads to the productionof multiple daughter RNAs. These daughter RNAs, as well as collinearsubgenomic transcripts, may be translated themselves to provide in situexpression of an encoded antigen, or may be transcribed to providefurther transcripts with the same sense as the delivered RNA which aretranslated to provide in situ expression of the antigen. The overallresult of this sequence of transcriptions is a huge amplification in thenumber of the introduced replicon RNAs and so the encoded antigenbecomes a major polypeptide product of the cells. Cells transfected withself-replicating RNA briefly produce antigen before undergoing apoptoticdeath. This death is a likely result of requisite double-stranded (ds)RNA intermediates, which also have been shown to super-activateDendritic Cells. Thus, the enhanced immunogenicity of self-replicatingRNA may be a result of the production of pro-inflammatory dsRNA, whichmimics an RNA-virus infection of host cells.

Advantageously, the cell's machinery is used by self-replicating RNAmolecules to generate an exponential increase of encoded gene products,such as proteins or antigens, which can accumulate in the cells or besecreted from the cells. Overexpression of proteins by self-replicatingRNA molecules takes advantage of the immunostimulatory adjuvant effects,including stimulation of toll-like receptors (TLR) 3, 7 and 8 and nonTLR pathways (e.g, RIG-1, MD-5) by the products of RNA replication andamplification, and translation which induces apoptosis of thetransfected cell.

The self-replicating RNA generally contains at least one or more genesselected from the group consisting of viral replicases, viral proteases,viral helicases and other nonstructural viral proteins, and alsocomprise 5′- and 3′-end cis-active replication sequences, and ifdesired, a heterologous sequences that encode a desired amino acidsequences (e.g., an antigen of interest). A subgenomic promoter thatdirects expression of the heterologous sequence can be included in theself-replicating RNA. If desired, the heterologous sequence (e.g., anantigen of interest) may be fused in frame to other coding regions inthe self-replicating RNA and/or may be under the control of an internalribosome entry site (IRES).

In certain embodiments, the self-replicating RNA molecule is notencapsulated in a virus-like particle. Self-replicating RNA molecules ofthe invention can be designed so that the self-replicating RNA moleculecannot induce production of infectious viral particles. This can beachieved, for example, by omitting one or more viral genes encodingstructural proteins that are necessary for the production of viralparticles in the self-replicating RNA. For example, when theself-replicating RNA molecule is based on an alpha virus, such asSinebis virus (SIN), Semliki forest virus and Venezuelan equineencephalitis virus (VEE), one or more genes encoding viral structuralproteins, such as capsid and/or envelope glycoproteins, can be omitted.

If desired, self-replicating RNA molecules of the invention can also bedesigned to induce production of infectious viral particles that areattenuated or virulent, or to produce viral particles that are capableof a single round of subsequent infection.

One suitable system for achieving self-replication in this manner is touse an alphavirus-based replicon. Alphaviruses comprise a set ofgenetically, structurally, and serologically related arthropod-borneviruses of the Togaviridae family. Twenty-six known viruses and virussubtypes have been classified within the alphavirus genus, including,Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelanequine encephalitis virus. As such, the self-replicating RNA of theinvention may incorporate a RNA replicase derived from semliki forestvirus (SFV), sindbis virus (SIN), Venezuelan equine encephalitis virus(VEE), Ross-River virus (RRV), eastern equine encephalitis virus, orother viruses belonging to the alphavirus family.

An alphavirus-based “replicon” expression vector can be used in theinvention. Replicon vectors may be utilized in several formats,including DNA, RNA, and recombinant replicon particles. Such repliconvectors have been derived from alphaviruses that include, for example,Sindbis virus (Xiong et al. (1989) Science 243:1188-1191; Dubensky etal., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol.72:950-958; Polo et al. (1999) PNAS 96:4598-4603), Semliki Forest virus(Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998)Nat. Biotech. 16:562-565), and Venezuelan equine encephalitis virus(Pushko et al. (1997) Virology 239:389-401). Alphavirus-derivedreplicons are generally quite similar in overall characteristics (e.g.,structure, replication), individual alphavirus may exhibit someparticular property (e.g., receptor binding, interferon sensitivity, anddisease profile) that is unique. Therefore, chimeric alphavirusreplicons made from divergent virus families may also be useful.

Alphavirus-based RNA replicons are typically (+)-stranded RNAs whichlead to translation of a replicase (or replicase-transcriptase) afterdelivery to a cell. The replicase is translated as a polyprotein whichauto-cleaves to provide a replication complex which creates genomic(−)-strand copies of the (+)-strand delivered RNA. These (−)-strandtranscripts can themselves be transcribed to give further copies of the(+)-stranded parent RNA and also to give a subgenomic transcript whichencodes the antigen. Translation of the subgenomic transcript thus leadsto in situ expression of the antigen by the infected cell. Suitablealphavirus replicons can use a replicase from a Sindbis virus, a Semlikiforest virus, an eastern equine encephalitis virus, a Venezuelan equineencephalitis virus, etc.

An RNA replicon preferably comprises an RNA genome from a picornavirus,togavirus, flavivirus, coronavirus, paramyxovirus, yellow fever virus,or alphavirus (e.g., Sindbis virus, Semliki Forest virus, Venezuelanequine encephalitis virus, or Ross River virus), which has been modifiedby the replacement of one or more structural protein genes with aselected heterologous nucleic acid sequence encoding a product ofinterest.

A preferred replicon encodes (i) a RNA-dependent RNA polymerase whichcan transcribe RNA from the replicon and (ii) an antigen. The polymerasecan be an alphavirus replicase e.g. comprising one or more of alphavirusproteins nsP1, nsP2, nsP3 and nsP4. Whereas natural alphavirus genomesencode structural virion proteins in addition to the non-structuralreplicase polyprotein, it is preferred that the replicon does not encodealphavirus structural proteins. Thus a preferred replicon can lead tothe production of genomic RNA copies of itself in a cell, but not to theproduction of RNA-containing virions. The inability to produce thesevirions means that, unlike a wild-type alphavirus, the preferredreplicon cannot perpetuate itself in infectious form. The alphavirusstructural proteins which are necessary for perpetuation in wild-typeviruses are absent from the preferred replicon and their place is takenby gene(s) encoding the antigen of interest, such that the subgenomictranscript encodes the antigen rather than the structural alphavirusvirion proteins.

A replicon useful with the invention may have two open reading frames.The first (5′) open reading frame encodes a replicase; the second (3′)open reading frame encodes an antigen. In some embodiments the RNA mayhave additional (e.g. downstream) open reading frames e.g. to encodeadditional antigens or to encode accessory polypeptides.

A preferred replicon has a 5′ cap (e.g. a 7-methylguanosine), whichoften can enhance in vivo translation of the RNA. In some embodimentsthe 5′ sequence of the replicon may need to be selected to ensurecompatibility with the encoded replicase.

A replicon may have a 3′ poly-A tail. It may also include a poly-Apolymerase recognition sequence (e.g. AAUAAA) near its 3′ end.

Replicons can have various lengths but they are typically 5000-25000nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides.

The replicon can conveniently be prepared by in vitro transcription(IVT). IVT can use a (cDNA) template created and propagated in plasmidform in bacteria, or created synthetically (for example by genesynthesis and/or polymerase chain-reaction (PCR) engineering methods).For instance, a DNA-dependent RNA polymerase (such as the bacteriophageT7, T3 or SP6 RNA polymerases) can be used to transcribe the repliconfrom a DNA template. Appropriate capping and poly-A addition reactionscan be used as required (although the replicon's poly-A is usuallyencoded within the DNA template). These RNA polymerases can havestringent requirements for the transcribed 5′ nucleotide(s) and in someembodiments these requirements must be matched with the requirements ofthe encoded replicase, to ensure that the IVT-transcribed RNA canfunction efficiently as a substrate for its self-encoded replicase.Specific examples include Sindbis-virus-based plasmids (pSIN) such aspSINCP, described, for example, in U.S. Pat. Nos. 5,814,482 and6,015,686, as well as in International Publication Nos. WO 97/38087, WO99/18226 and WO 02/26209. The construction of such replicons, ingeneral, is described in U.S. Pat. Nos. 5,814,482 and 6,015,686.

In other aspects, the self-replicating RNA molecule is derived from orbased on a virus other than an alphavirus, preferably, apositive-stranded RNA virus, and more preferably a picornavirus,flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, orcoronavirus. Suitable wild-type alphavirus sequences are well-known andare available from sequence depositories, such as the American TypeCulture Collection, Rockville, Md. Representative examples of suitablealphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCCVR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCCVR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCCVR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCCVR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Mayaro virus(ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580,ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCCVR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest(ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR-1248),Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374),Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCCVR-1250 ATCC VR-1249, ATCC VR-532), Western equine encephalomyelitis(ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCCVR-926), and Y-62-33 (ATCC VR-375).

The self-replicating RNA molecules of the invention are larger thanother types of RNA (e.g. mRNA) that have been prepared using modifiednucleotides. Typically, the self-replicating RNA molecules of theinvention contain at least about 4 kb. For example, the self-replicatingRNA can contain at least about 5 kb, at least about 6 kb, at least about7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, atleast about 11 kb, at least about 12 kb or more than 12 kb. In certainexamples, the self-replicating RNA is about 4 kb to about 12 kb, about 5kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb,about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb toabout 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb,about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb toabout 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb,about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb toabout 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb,about 9 kb to about 10 kb, or about 10 kb to about 11 kb.

The self-replicating RNA molecules of the invention may comprise one ormore types of modified nucleotides (e.g., pseudouridine,N6-methyladenosine, 5-methylcytidine, 5-methyluridine).

The self-replicating RNA molecule may encode a single heterologouspolypeptide antigen or, optionally, two or more heterologous polypeptideantigens linked together in a way that each of the sequences retains itsidentity (e.g., linked in series) when expressed as an amino acidsequence. The heterologous polypeptides generated from theself-replicating RNA may then be produced as a fusion polypeptide orengineered in such a manner to result in separate polypeptide or peptidesequences.

The self-replicating RNA of the invention may encode one or morepolypeptide antigens that contain a range of epitopes. Preferablyepitopes capable of eliciting either a helper T-cell response or acytotoxic T-cell response or both.

The self-replicating RNA molecules described herein may be engineered toexpress multiple nucleotide sequences, from two or more open readingframes, thereby allowing co-expression of proteins, such as two or moreantigens together with cytokines or other immunomodulators, which canenhance the generation of an immune response. Such a self-replicatingRNA molecule might be particularly useful, for example, in theproduction of various gene products (e.g., proteins) at the same time,for example, as a bivalent or multivalent vaccine.

The self-replicating RNA molecules of the invention can be preparedusing any suitable method. Several suitable methods are known in the artfor producing RNA molecules that contain modified nucleotides. Forexample, a self-replicating RNA molecule that contains modifiednucleotides can be prepared by transcribing (e.g., in vitrotranscription) a DNA that encodes the self-replicating RNA moleculeusing a suitable DNA-dependent RNA polymerase, such as T7 phage RNApolymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, and thelike, or mutants of these polymerases which allow efficientincorporation of modified nucleotides into RNA molecules. Thetranscription reaction will contain nucleotides and modifiednucleotides, and other components that support the activity of theselected polymerase, such as a suitable buffer, and suitable salts. Theincorporation of nucleotide analogs into a self-replicating RNA may beengineered, for example, to alter the stability of such RNA molecules,to increase resistance against RNases, to establish replication afterintroduction into appropriate host cells (“infectivity” of the RNA),and/or to induce or reduce innate and adaptive immune responses.

Suitable synthetic methods can be used alone, or in combination with oneor more other methods (e.g., recombinant DNA or RNA technology), toproduce a self-replicating RNA molecule of the invention. Suitablemethods for de novo synthesis are well-known in the art and can beadapted for particular applications. Exemplary methods include, forexample, chemical synthesis using suitable protecting groups such as CEM(Masuda et al., (2007) Nucleic Acids Symposium Series 51:3-4), theβ-cyanoethyl phosphoramidite method (Beaucage S L et al. (1981)Tetrahedron Lett 22:1859); nucleoside H-phosphonate method (Garegg P etal. (1986) Tetrahedron Lett 27:4051-4; Froehler B C et al. (1986) NuclAcid Res 14:5399-407; Garegg P et al. (1986) Tetrahedron Lett 27:4055-8;Gaffney B L et al. (1988) Tetrahedron Lett 29:2619-22). Thesechemistries can be performed or adapted for use with automated nucleicacid synthesizers that are commercially available. Additional suitablesynthetic methods are disclosed in Uhlmann et al. (1990) Chem Rev90:544-84, and Goodchild J (1990) Bioconjugate Chem 1: 165. Nucleic acidsynthesis can also be performed using suitable recombinant methods thatare well-known and conventional in the art, including cloning,processing, and/or expression of polynucleotides and gene productsencoded by such polynucleotides. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic polynucleotides areexamples of known techniques that can be used to design and engineerpolynucleotide sequences. Site-directed mutagenesis can be used to alternucleic acids and the encoded proteins, for example, to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and the like.Suitable methods for transcription, translation and expression ofnucleic acid sequences are known and conventional in the art. (Seegenerally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel,et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988;Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986;Bitter, et al., in Methods in Enzymology 153:516-544 (1987); TheMolecular Biology of the Yeast Saccharomyces, Eds. Strathern et al.,Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

The presence and/or quantity of one or more modified nucleotides in aself-replicating RNA molecule can be determined using any suitablemethod. For example, a self-replicating RNA can be digested tomonophosphates (e.g., using nuclease P1) and dephosphorylated (e.g.,using a suitable phosphatase such as CIAP), and the resultingnucleosides analyzed by reversed phase HPLC (e.g., usings a YMC PackODS-AQ column (5 micron, 4.6×250 mm) and elute using a gradient, 30% B(0-5 min) to 100% B (5-13 min) and at 100% B (13-40) min, flow Rate (0.7ml/min), UV detection (wavelength: 260 nm), column temperature (30° C.).Buffer A (20 mM acetic acid—ammonium acetate pH 3.5), buffer B (20 mMacetic acid—ammonium acetate pH 3.5/methanol [90/10])).

Optionally, the self-replicating RNA molecules of the invention mayinclude one or more modified nucleotides so that the self-replicatingRNA molecule will have less immunomodulatory activity upon introductionor entry into a host cell (e.g., a human cell) in comparison to thecorresponding self-replicating RNA molecule that does not containmodified nucleotides.

If desired, the self-replicating RNA molecules can be screened oranalyzed to confirm their therapeutic and prophylactic properties usingvarious in vitro or in vivo testing methods that are known to those ofskill in the art. For example, vaccines comprising self-replicating RNAmolecule can be tested for their effect on induction of proliferation oreffector function of the particular lymphocyte type of interest, e.g., Bcells, T cells, T cell lines, and T cell clones. For example, spleencells from immunized mice can be isolated and the capacity of cytotoxicT lymphocytes to lyse autologous target cells that contain a selfreplicating RNA molecule that encodes a polypeptide antigen. Inaddition, T helper cell differentiation can be analyzed by measuringproliferation or production of TH1 (IL-2 and IFN-γ) and/or TH2 (IL-4 andIL-5) cytokines by ELISA or directly in CD4+ T cells by cytoplasmiccytokine staining and flow cytometry.

Self-replicating RNA molecules that encode a polypeptide antigen canalso be tested for ability to induce humoral immune responses, asevidenced, for example, by induction of B cell production of antibodiesspecific for an antigen of interest. These assays can be conductedusing, for example, peripheral B lymphocytes from immunized individuals.Such assay methods are known to those of skill in the art. Other assaysthat can be used to characterize the self-replicating RNA molecules ofthe invention can involve detecting expression of the encoded antigen bythe target cells. For example, FACS can be used to detect antigenexpression on the cell surface or intracellularly. Another advantage ofFACS selection is that one can sort for different levels of expression;sometimes-lower expression may be desired. Other suitable method foridentifying cells which express a particular antigen involve panningusing monoclonal antibodies on a plate or capture using magnetic beadscoated with monoclonal antibodies.

B. Antigens

In certain embodiments, the nucleic acid molecule described herein is anucleic acid molecule (e.g., an RNA molecule) that encodes an antigen.Suitable antigens include, but are not limited to, a bacterial antigen,a viral antigen, a fungal antigen, a protazoan antigen, a plant antigen,a cancer antigen, or a combination thereof.

Suitable antigens include proteins and peptides from a pathogen such asa virus, bacteria, fungus, protozoan, plant or from a tumor. Viralantigens and immunogens that can be encoded by the self-replicating RNAmolecule include, but are not limited to, proteins and peptides from aOrthomyxoviruses, such as Influenza A, B and C; Paramyxoviridae viruses,such as Pneumoviruses (RSV), Paramyxoviruses (PIV), Metapneumovirus andMorbilliviruses (e.g., measles); Pneumoviruses, such as Respiratorysyncytial virus (RSV), Bovine respiratory syncytial virus, Pneumoniavirus of mice, and Turkey rhinotracheitis virus; Paramyxoviruses, suchas Parainfluenza virus types 1-4 (PIV), Mumps virus, Sendai viruses,Simian virus 5, Bovine parainfluenza virus, Nipahvirus, Henipavirus andNewcastle disease virus; Poxviridae, including a Orthopoxvirus such asVariola vera (including but not limited to, Variola major and Variolaminor); Metapneumoviruses, such as human metapneumovirus (hMPV) andavian metapneumoviruses (aMPV); Morbilliviruses, such as Measles;Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus,Parechovirus, Cardioviruses and Aphthoviruses; Enteroviruseses, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71, Bunyaviruses, including aOrthobunyavirus such as California encephalitis virus; a Phlebovirus,such as Rift Valley Fever virus; a Nairovirus, such as Crimean-Congohemorrhagic fever virus; Heparnaviruses, such as, Hepatitis A virus(HAV); Togaviruses (Rubella), such as a Rubivirus, an Alphavirus, or anArterivirus; Flaviviruses, such as Tick-borne encephalitis (TBE) virus,Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japaneseencephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus,St. Louis encephalitis virus, Russian spring-summer encephalitis virus,Powassan encephalitis virus; Pestiviruses, such as Bovine viral diarrhea(BVDV), Classical swine fever (CSFV) or Border disease (BDV);Hepadnaviruses, such as Hepatitis B virus, Hepatitis C virus;Rhabdoviruses, such as a Lyssavirus (Rabies virus) and Vesiculovirus(VSV), Caliciviridae, such as Norwalk virus, and Norwalk-like Viruses,such as Hawaii Virus and Snow Mountain Virus; Coronaviruses, such asSARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV),Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritisvirus (TGEV); Retroviruses such as an Oncovirus, a Lentivirus or aSpumavirus; Reoviruses, as an Orthoreovirus, a Rotavirus, an Orbivirus,or a Coltivirus; Parvoviruses, such as Parvovirus B19; Delta hepatitisvirus (HDV); Hepatitis E virus (HEV); Hepatitis G virus (HGV); HumanHerpesviruses, such as, by way Herpes Simplex Viruses (HSV),Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus(CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and HumanHerpesvirus 8 (HHV8); Papovaviruses, such as Papillomaviruses andPolyomaviruses, Adenoviruess and Arenaviruses.

In some embodiments, the antigen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

In some embodiments the antigen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunizingagainst malaria. In some embodiments the antigen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

Bacterial antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Neisseria meningitides, Streptococcus pneumoniae,Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis,Burkholderia sp. (e.g., Burkholderia mallei, Burkholderia pseudomalleiand Burkholderia cepacia), Staphylococcus aureus, Staphylococcusepidermis, Haemophilus influenzae, Clostridium tetani (Tetanus),Clostridium perfringens, Clostridium botulinums (Botulism),Cornynebacterium diphtheriae (Diphtheria), Pseudomonas aeruginosa,Legionella pneumophila, Coxiella burnetii, Brucella sp. (e.g., B.abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis and B.pinnipediae), Francisella sp. (e.g., F. novicida, F. philomiragia and F.tularensis), Streptococcus agalactiae, Neiserria gonorrhoeae, Chlamydiatrachomatis, Treponema pallidum (Syphilis), Haemophilus ducreyi,Enterococcus faecalis, Enterococcus faecium, Helicobacter pylori,Staphylococcus saprophyticus, Yersinia enterocolitica, E. coli (such asenterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC),diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC),extraintestinal pathogenic E. coli (ExPEC; such as uropathogenic E. coli(UPEC) and meningitis/sepsis-associated E. coli (MNEC)), and/orenterohemorrhagic E. coli (EHEC), Bacillus anthracis (anthrax), Yersiniapestis (plague), Mycobacterium tuberculosis, Rickettsia, Listeriamonocytogenes, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi(typhoid fever), Borrelia burgdorfer, Porphyromonas gingivalis,Klebsiella, Mycoplasma pneumoniae, etc.

Fungal antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Dermatophytres, including: Epidermophyton floccusum,Microsporum audouini, Microsporum canis, Microsporum distortum,Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophytonconcentricum, Trichophyton equinum, Trichophyton gallinae, Trichophytongypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophytonquinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophytontonsurans, Trichophyton verrucosum, T. verrucosum var. album, var.discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophytonfaviforme; or from Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowii, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septataintestinalis and Enterocytozoon bieneusi; the less common are Brachiolaspp, Microsporidium spp., Nosema spp., Pleistophora spp.,Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis,Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale,Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii,Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaeaspp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolusspp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp,Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp,Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp,Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, andCladosporium spp.

Protazoan antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Entamoeba histolytica, Giardia lambli, Cryptosporidiumparvum, Cyclospora cayatanensis and Toxoplasma.

Plant antigens and immunogens that can be encoded by theself-replicating RNA molecule include, but are not limited to, proteinsand peptides from Ricinus communis.

Suitable antigens include proteins and peptides from a virus such as,for example, human immunodeficiency virus (HIV), hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplexvirus (HSV), cytomegalovirus (CMV), influenza virus (flu), respiratorysyncytial virus (RSV), parvovorus, norovirus, human papilloma virus(HPV), rhinovirus, yellow fever virus, rabies virus, Dengue fever virus,measles virus, mumps virus, rubella virus, varicella zoster virus,enterovirus (e.g., enterovirus 71), ebola virus, and bovine diarrheavirus. Preferably, the antigenic substance is selected from the groupconsisting of HSV glycoprotein gD, HIV glycoprotein gp120, HIVglycoprotein gp 40, HIV p55 gag, and polypeptides from the pol and tatregions. In other preferred embodiments of the invention, the antigen isa protein or peptide derived from a bacterium such as, for example,Helicobacter pylori, Haemophilus influenza, Vibrio cholerae (cholera),C. diphtheriae (diphtheria), C. tetani (tetanus), Neisseriameningitidis, B. pertussis, Mycobacterium tuberculosis, and the like.

HIV antigens that can be encoded by the self-replicating RNA moleculesof the invention are described in U.S. application Ser. No. 490,858,filed Mar. 9, 1990, and published European application number 181150(May 14, 1986), as well as U.S. application Ser. Nos. 60/168,471;09/475,515; 09/475,504; and Ser. No. 09/610,313, the disclosures ofwhich are incorporated herein by reference in their entirety.

Cytomegalovirus antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in U.S. Pat. No. 4,689,225,U.S. application Ser. No. 367,363, filed Jun. 16, 1989 and PCTPublication WO 89/07143, the disclosures of which are incorporatedherein by reference in their entirety.

Hepatitis C antigens that can be encoded by the self-replicating RNAmolecules of the invention are described in PCT/US88/04125, publishedEuropean application number 318216 (May 31, 1989), published Japaneseapplication number 1-500565 filed Nov. 18, 1988, Canadian application583,561, and EPO 388,232, disclosures of which are incorporated hereinby reference in their entirety. A different set of HCV antigens isdescribed in European patent application 90/302866.0, filed Mar. 16,1990, and U.S. application Ser. No. 456,637, filed Dec. 21, 1989, andPCT/US90/01348, the disclosures of which are incorporated herein byreference in their entirety.

In some embodiments, the antigen is derived from an allergen, such aspollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In certain embodiments, a tumor immunogen or antigen, or cancerimmunogen or antigen, can be encoded by the self-replicating RNAmolecule. In certain embodiments, the tumor immunogens and antigens arepeptide-containing tumor antigens, such as a polypeptide tumor antigenor glycoprotein tumor antigens.

Tumor immunogens and antigens appropriate for the use herein encompass awide variety of molecules, such as (a) polypeptide-containing tumorantigens, including polypeptides (which can range, for example, from8-20 amino acids in length, although lengths outside this range are alsocommon), lipopolypeptides and glycoproteins.

In certain embodiments, tumor immunogens are, for example, (a) fulllength molecules associated with cancer cells, (b) homologs and modifiedforms of the same, including molecules with deleted, added and/orsubstituted portions, and (c) fragments of the same. Tumor immunogensinclude, for example, class I-restricted antigens recognized by CD8+lymphocytes or class II-restricted antigens recognized by CD4+lymphocytes.

In certain embodiments, tumor immunogens include, but are not limitedto, (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well asRAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1,GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12(which can be used, for example, to address melanoma, lung, head andneck, NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutatedantigens, for example, p53 (associated with various solid tumors, e.g.,colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g.,melanoma, pancreatic cancer and colorectal cancer), CDK4 (associatedwith, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8(associated with, e.g., head and neck cancer), CIA 0205 (associatedwith, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associatedwith, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkinslymphoma), BCR-abl (associated with, e.g., chronic myelogenousleukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT,(c) over-expressed antigens, for example, Galectin 4 (associated with,e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA(associated with, e.g., colorectal cancer), gastrin (associated with,e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e.g., breast and ovarian cancer), G-250(associated with, e.g., renal cell carcinoma), p53 (associated with,e.g., breast, colon cancer), and carcinoembryonic antigen (associatedwith, e.g., breast cancer, lung cancer, and cancers of thegastrointestinal tract such as colorectal cancer), (d) shared antigens,for example, melanoma-melanocyte differentiation antigens such asMART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example).

In certain embodiments, tumor immunogens include, but are not limitedto, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EpsteinBarr virus antigens, EBNA, human papillomavirus (HPV) antigens,including E6 and E7, hepatitis B and C virus antigens, human T-celllymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met,mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE,PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029,FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

C. Aqueous Solution for the Nucleic Acid Molecule

The nucleic acid molecule (such as RNA) is generally provided in theform of an aqueous solution, or a form that can be readily dissolved inan aqueous solution (e.g., lyophilized). The aqueous solution can be inwater, or an aqueous solution that comprises a salt (e.g., NaCl), abuffer (e.g., a citrate buffer), a nonionic tonicifying agent (e.g., asaccharide), a polymer, a surfactant, or a combination thereof. If theformulation is intended for in vivo administration, it is preferablethat the aqueous solution is a physiologically acceptable buffer thatmaintains a pH that is compatible with normal physiological conditions.Also, in certain instances, it may be desirable to maintain the pH at aparticular level in order to insure the stability of certain componentsof the formulation.

For example, it may be desirable to prepare an aqueous solution that isisotonic and/or isosmotic. Hypertonic and hypotonic solutions sometimescould cause complications and undesirable effects when injected, such aspost-administration swelling or rapid absorption of the compositionbecause of differential ion concentrations between the composition andphysiological fluids. To control tonicity, the emulsion may comprise aphysiological salt, such as a sodium salt. Sodium chloride (NaCl), forexample, may be used at about 0.9% (w/v) (physiological saline). Othersalts that may be present include potassium chloride, potassiumdihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride,calcium chloride, etc. In an exemplary embodiment, the aqueous solutioncomprises 10 mM NaCl and other salts or non-ionic tonicifying agents. Asdescribed herein, non-ionic tonicifying agents can also be used tocontrol tonicity. In an embodiment, hypertonic and hypotonic solutionsare mixed (e.g., hypertonic RNA an hypotonic emulsion; hypotonic RNA andhypertonic emulsion) such that once mixed they form an isotonicsolution.

The aqueous solution may be buffered. Any physiologically acceptablebuffer may be used herein, such as citrate buffers, phosphate buffers,acetate buffers, succinate buffer, tris buffers, bicarbonate buffers,carbonate buffers, or the like. The pH of the aqueous solution willpreferably be between 6.0-8.0, preferably about 6.2 to about 6.8. Insome cases, certain amount of salt may be included in the buffer. Inother cases, salt in the buffer might interfere with complexation ofnucleic acid molecules to the emulsion particle, therefore is avoided.

The aqueous solution may also comprise additional components such asmolecules that change the osmolarity of the aqueous solution ormolecules that stabilizes the nucleic acid molecule after complexation.For example, the osmolality can be adjusted using a non-ionictonicifying agent, which are generally carbohydrates but can also bepolymers. (See, e.g., Voet and Voet (1990) Biochemistry (John Wiley &Sons, New York.) Examples of suitable non-ionic tonicifying agentsinclude sugars (e.g., trehalose, sucrose, dextrose, fructose, reducedpalatinose, etc.), sugar alcohols (such as mannitol, sorbitol, xylitol,erythritol, lactitol, maltitol, glycerol, etc.), and combinationsthereof. If desired, a nonionic polymer (e.g., a poly(alkyl glycol) suchas polyethylene glycol, polypropylene glycol, or polybutlyene glycol) ornonionic surfactant can be used. These types of agents, in particularsugar and sugar alcohols, are also cryoprotectants that can procted RNA,and other nucleic acid molecules, when lyophilized. In exemplaryembodiments, the buffer comprises from about 560 nM to 600 mM oftrehalose, sucrose, sorbitol, or dextrose.

In some cases, it may be preferable to prepare an aqueous solutioncomprising the nucleic acid molecule as a hypertonic solution, and toprepare the cationic emulsion using unadulterated water or a hypotonicbuffer. When the emulsion and the nucleic acid molecule are combined,the mixture becomes isotonic. For example, an aqueous solutioncomprising RNA can be a 2× hypertonic solution, and the cationicemulsion can be prepared using 10 mM Citrate buffer. When the RNAsolution and the emulsion are mixed at 1:1 (v/v) ratio, the compositionbecomes isotonic. Based on desired relative amounts of the emulsion tothe aqueous solution that comprises the nucleic acid molecule (e.g., 1:1(v/v) mix, 2:1 (v/v) mix, 1:2 (v/v) mix, etc.), one can readilydetermine the tonicity of the aqueous solution that is required in orderto achieve an isotonic mixture.

Similarly, compositions that have physiological osmolality may bedesirable for in vivo administration. Physiological osmolality is fromabout 255 mOsm/kg water to about 315 mOsm/kg water. Sometimes, it may bepreferable to prepare an aqueous solution comprising the nucleic acidmolecule as a hyperosmolar solution, and to prepare the cationicemulsion using unadulterated water or a hypoosmolar buffer. When theemulsion and the nucleic acid molecule are combined, physiologicalosmolality is achieved. Of course, this can also be achieved using ahypoosmolar nucleic acid solution and a hyperosmolar buffer. Based ondesired relative amounts of the emulsion to the aqueous solution thatcomprises the nucleic acid molecule (e.g., 1:1 (v/v) mix, 2:1 (v/v) mix,1:2 (v/v) mix, etc.), one can readily determine the osmolality of theaqueous solution that is required in order to achieve an iso-osmolarmixture.

In certain embodiments, the aqueous solution comprising the nucleic acidmolecule may further comprise a polymer or a surfactant, or acombination thereof. In an exemplary embodiment, the oil-in-wateremulsion contains a poloxamer. In particular, the inventors haveobserved that adding Pluronic® F127 to the RNA aqueous solution prior tocomplexation to cationic emulsion particles led to greater stability andincreased RNase resistance of the RNA molecule. Addition of pluronicF127 to RNA aqueous solution was also found to decrease the particlesize of the RNA/CNE complex. Poloxamer polymers may also facilitateappropriate decomplexation/release of the RNA molecule, preventaggregation of the emulsion particles, and have immune modulatoryeffect. Other polymers that may be used include, e.g., Pluronic® F68 orPEG300.

Alternatively or in addition, the aqueous solution comprising thenucleic acid molecule may comprise from about 0.05% to about 20% (w/v)polymer. For example, the cationic oil-in-water emulsion may comprise apolymer (e.g., a poloxamer such as Pluronic® F127, Pluronic® F68, orPEG300) at from about 0.05% to about 10% (w/v), such as 0.05%, 0.5%, 1%,or 5%.

The buffer system may comprise any combination of two or more moleculesdescribed above (salt, buffer, saccharide, polymer, etc). In anpreferred embodiment, the buffer comprises 560 mM sucrose, 20 mM NaCl,and 2 mM Citrate, which can be mixed with a cationic oil in wateremulsion described herein to produce a final aqueous phase thatcomprises 280 mM sucrose, 10 mM NaCl and 1 mM citrate. In otherembodiments, the buffer comprises about 2-20 mM Citrate, which can bemixed with a cationic oil in water emulsion described herein to producea final aqueous phase that comprises about 1-10 mM Citrate.

5. Methods of Preparation

In another aspect, the invention provides a method of preparing acomposition that comprises a nucleic acid molecule complexed with aparticle of a cationic oil-in-water emulsion at an N/P ratio of at least4:1 and with average particle diameter from about 80 nm to about 180 nm,comprising: preparing a cationic oil-in-water emulsion wherein theemulsion comprises: (1) from about 0.2% to about 20% (v/v) oil, (2) fromabout 0.01% to about 2.5% (v/v) surfactant, and (3) a cationic lipid;and adding the nucleic acid molecule to the cationic oil-in-wateremulsion so that the nucleic acid molecule complexes with the particleof the emulsion.

One exemplary approach to generate the cationic oil-in-water emulsion isby a process comprising: (1) combining the oil and the cationic lipid toform the oil phase of the emulsion; (2) providing an aqueous solution toform the aqueous phase of the emulsion; and (3) dispersing the oil phasein the aqueous phase, for example, by homogenization. Homogenization maybe achieved in any suitable way, for example, using a commercialhomogenizer (e.g., IKA T25 homogenizer, available at VWR International(West Chester, Pa.).

The cationic lipid may be dissolved in a suitable solvent, such aschloroform (CHCl₃), dichloromethane (DCM), ethanol, acetone,Tetrahydrofuran (THF), 2,2,2 trifluoroethanol, acetonitrile, ethylacetate, hexane, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO),etc., and added directly to the oil component of the emulsion.Alternatively, the cationic lipid may be added to a suitable solvent toform a liposome suspension; then the liposome suspension may be added tothe oil component of the emulsion. The cationic lipid may also bedissolved directly in the oil.

It may be desirable to heat the oil to a temperature between about 30°C. to about 65° C. to facilitate the dissolving of the lipid.

Desired amount of the cationic lipid (e.g., DOTAP) can be measured andeither dissolved in a solvent, in water, or directly in oil to reach adesired final concentration as described and exemplified herein.

Solvents such as chloroform (CHCl₃) or dichloromethane (DCM) may beremoved from the oil phase, e.g., by evaporation, prior to combining theaqueous phase and the oil phase or prior to homogenization.Alternatively, in instances where lipid solubility can be an issue, aprimary emulsion can be made with the solvent (e.g. DCM) still in theoil phase. In such cases, the solvent can be removed (e.g., allowed toevaporate) from the primary emulsion prior to a secondaryhomogenization.

If the emulsion comprises one or more surfactants, the surfactant(s) maybe included in the oil phase or the aqueous phase according to theconventional practice in the art. For example, SPAN®85 can be dissolvedin the oil phase (e.g., squalene), and Tween® 80 may be dissolved in theaqueous phase (e.g., in a citrate buffer).

In another aspect, the invention provides a method of preparing acomposition that comprises a nucleic acid molecule (such as RNA)complexed with a particle of a cationic oil-in-water emulsion,comprising: (i) providing a cationic oil-in-water emulsion as describedherein; (ii) providing a aqueous solution comprising the nucleic acidmolecule (such as RNA); and (iii) combining the oil-in-water emulsion of(i) and the aqueous solution of (iii), so that the nucleic acid moleculecomplexes with the particle of the emulsion. For example, a cationicoil-in-water emulsion may be combined with an aqueous solutioncomprising a nucleic acid molecule (e.g., an aqueous RNA solution) inany desired relative amounts, e.g., about 1:1 (v/v), about 1.5:1 (v/v),about 2:1 (v/v), about 2.5:1 (v/v), about 3:1 (v/v), about 3.5:1 (v/v),about 4:1 (v/v), about 5:1 (v/v), about 10:1 (v/v), about 1:1.5 (v/v),about 1:2 (v/v), about 1:2.5 (v/v), about 1:3 (v/v), about 1:3.5 (v/v),about 1:4 (v/v), about 1:1.5 (v/v), or about 1:1.10 (v/v), etc.

The concentration of each component of the post-complex composition(e.g., RNA-emulsion complex) can be readily determined according torelative amounts of the pre-complex oil-in-water emulsion and theaqueous solution comprising the nucleic acid molecule (e.g., an aqueousRNA solution) that are used. For example, when a cationic oil-in-wateremulsion is combined with an aqueous solution comprising a nucleic acidmolecule (e.g., an aqueous RNA solution) at 1:1 (v:v) ratio, theconcentrations of the oil and cationic lipid become ½ of that of thepre-complex emulsion. Therefore, if an emulsion comprising 4.3% (w/v)squalene, 1.4 mg/mL DOTAP, 0.5% v/v SPAN®85 and 0.5% v/v Tween® 80(referred herein as “CNE17”) is combined with an aqueous RNA solutionthat comprises 560 mM sucrose, 20 mM NaCl, 2 mM Citrate, and 1% (w/v)Pluronic F127 at 1:1 (v:v), the post-complex composition comprises 2.15%(w/v) squalene, 0.7 mg/mL DOTAP, 0.25% v/v SPAN®85, 0.25% v/v Tween® 80,280 mM sucrose, 10 mM NaCl, 1 mM Citrate, and 0.5% (w/v) Pluronic F127.

Additional optional steps to promote particle formation, to improve thecomplexation between the nucleic acid molecules and the cationicparticles, to increase the stability of the nucleic acid molecule (e.g.,to prevent degradation of an RNA molecule), to facilitate appropriatedecomplexation/release of the nucleic acid molecules (such as an RNAmolecule), or to prevent aggregation of the emulsion particles may beincluded. For example, a polymer (e.g., Pluronic® F127) or a surfactantmay be added to the aqueous solution that comprises the nucleic acidmolecule (such as RNA). In one exemplary embodiment, Pluronic® F127 isadded to the RNA molecule prior to complexation to the emulsionparticle.

The size of the emulsion particles can be varied by changing the ratioof surfactant to oil (increasing the ratio decreases droplet size),operating pressure (increasing operating pressure reduces droplet size),temperature (increasing temperature decreases droplet size), and otherprocess parameters. Actual particle size will also vary with theparticular surfactant, oil, and cationic lipid used, and with theparticular operating conditions selected. Emulsion particle size can beverified by use of sizing instruments, such as the commercial Sub-MicronParticle Analyzer (Model N4MD) manufactured by the Coulter Corporation,and the parameters can be varied using the guidelines set forth aboveuntil the average diameter of the particles is 80 nm to 180 nm.

Optional processes for preparing the cationic oil-in-water emulsion(pre-complexation emulsion), or the nucleic acid molecule-emulsioncomplex, include, e.g., sterilization, particle size selection (e.g.,removing large particles), filling, packaging, and labeling, etc.

For example, if the pre-complexation emulsion, or the nucleic acidmolecule-emulsion complex, is formulated for in vivo administration, itmay be sterilized, e.g., by filtering through a sterilizing grade filter(e.g., through a 0.22 micron filter). Other sterilization techniquesinclude a thermal process, or a radiation sterilization process, orusing pulsed light to produce a sterile composition.

The cationic oil-in-water emulsion described herein can be used tomanufacture vaccines. Sterile and/or clinical grade cationicoil-in-water emulsions can be prepared using similar methods asdescribed for MF59. See, e.g., Ott et al., Methods in MolecularMedicine, 2000, Volume 42, 211-228, in VACCINE ADJUVANTS (O'Hagan ed.),Humana Press. For example, similar to the manufacturing process of MF59,the oil phase and the aqueous phase of the emulsion can be combined andprocessed in an inline homogenizer to yield a coarse emulsion. Thecoarse emulsion can then be fed into a microfluidizer, where it can befurther processed to obtain a stable submicron emulsion. The coarseemulsion can be passed through the interaction chamber of themicrofluidizer repeatedly until the desired particle size is obtained.The bulk emulsion can then be filtered (e.g., though a 0.22-μm filterunder nitrogen) to remove large particles, yielding emulsion bulk thatcan be filled into suitable containers (e.g., glass bottles). Forvaccine antigens that have demonstrated long-term stability in thepresence of oil-in-water emulsion for self storage, the antigen andemulsion may be combined and sterile-filtered (e.g., though a 0.22-μmfilter membrane). The combined single vial vaccine can be filled intosingle-dose containers. For vaccine antigens where long-term stabilityhas not been demonstrated, the emulsion can be supplied as a separatevial. In such cases, the emulsion bulk can be filtered-sterilized (e.g.,though a 0.22-μm filter membrane), filled, and packaged in finalsingle-dose vials.

Quality control may be optionally performed on a small sample of theemulsion bulk or admixed vaccine, and the bulk or admixed vaccine willbe packaged into doses only if the sample passes the quality controltest.

6. Pharmaceutical Compositions and Administration

In another aspect, the invention provides a pharmaceutical compositioncomprising a nucleic acid molecule complexed with a particle of acationic oil-in-water emulsion, as described herein, and may furthercomprise one or more pharmaceutically acceptable carriers, diluents, orexcipients. In preferred embodiments, the pharmaceutical composition isan immunogenic composition, which can be used as a vaccine.

The compositions described herein may be used to deliver a nucleic acidmolecule to cells. For example, nucleic acid molecules (e.g., DNA orRNA) can be delivered to cells for a variety of purposes, such as toinduce production of a desired gene product (e.g., protein), to regulateexpression of a gene, for gene therapy and the like. The compositionsdescribed herein may also be used to deliver a nucleic acid molecule(e.g., DNA or RNA) to cells for therapeutic purposes, such as to treat adisease such as cancers or proliferative disorders, metabolic diseases,cardiovascular diseases, infections, allergies, to induce an immuneresponse and the like. For example, nucleic acid molecules may bedelivered to cells to inhibit the expression of a target gene. Suchnucleic acid molecules include, e.g., antisense oligonucleotides,double-stranded RNAs, such as small interfering RNAs and the like.Double-stranded RNA molecules, such as small interfering RNAs, cantrigger RNA interference, which specifically silences the correspondingtarget gene (gene knock down). Antisense oligonucleotides are singlestrands of DNA or RNA that are complementary to a chosen sequence.Generally, antisense RNA can prevent protein translation of certainmessenger RNA strands by binding to them. Antisense DNA can be used totarget a specific, complementary (coding or non-coding) RNA. Therefore,the cationic emulsions described herein are useful for deliveringantisense oligonucleotides or double-stranded RNAs for treatment of, forexample, cancer by inhibiting production of an oncology target.

The pharmaceutical compositions provided herein may be administeredsingly or in combination with one or more additional therapeutic agents.The methods of administration include, but are not limited to, oraladministration, rectal administration, parenteral administration,subcutaneous administration, intravenous administration, intravitrealadministration, intramuscular administration, inhalation, intranasaladministration, topical administration, ophthalmic administration, andotic administration.

A therapeutically effective amount of the compositions described hereinwill vary depending on, among others, the disease indicated, theseverity of the disease, the age and relative health of the subject, thepotency of the compound administered, the mode of administration and thetreatment desired.

In other embodiments, the pharmaceutical compositions described hereincan be administered in combination with one or more additionaltherapeutic agents. The additional therapeutic agents may include, butare not limited to antibiotics, antibacterial agents, antiemetic agents,antifungal agents, anti-inflammatory agents, antiviral agents,immunomodulatory agents, cytokines, antidepressants, hormones,alkylating agents, antimetabolites, antitumour antibiotics, antimitoticagents, topoisomerase inhibitors, cytostatic agents, anti-invasionagents, antiangiogenic agents, inhibitors of growth factor functioninhibitors of viral replication, viral enzyme inhibitors, anticanceragents, α-interferons, β-interferons, ribavirin, hormones, othertoll-like receptor modulators, immunoglobulins (Igs), and antibodiesmodulating Ig function (such as anti-IgE (omalizumab)).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of infectious diseases including, but notlimited to, disease caused by the pathogens disclosed herein, includingviral diseases such as genital warts, common warts, plantar warts,rabies, respiratory syncytial virus (RSV), hepatitis B, hepatitis C,Dengue virus, yellow fever, herpes simplex virus (by way of exampleonly, HSV-I, HSV-II, CMV, or VZV), molluscum contagiosum, vaccinia,variola, lentivirus, human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis virus (hepatitis C virus, hepatitis B virus,hepatitis A virus), cytomegalovirus (CMV), varicella zoster virus (VZV),rhinovirus, enterovirus (e.g. EV71), adenovirus, coronavirus (e.g.,SARS), influenza, para-influenza, mumps virus, measles virus, rubellavirus, papovavirus, hepadnavirus, flavivirus, retrovirus, arenavirus (byway of example only, LCM, Junin virus, Machupo virus, Guanarito virusand Lassa Fever) and filovirus (by way of example only, ebola virus ormarburg virus).

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of bacterial, fungal, and protozoal infectionsincluding, but not limited to, malaria, tuberculosis and mycobacteriumavium, leprosy; pneumocystis carnii, cryptosporidiosis, histoplasmosis,toxoplasmosis, trypanosome infection, leishmaniasis, infections causedby bacteria of the genus Escherichia, Enterobacter, Salmonella,Staphylococcus, Klebsiella, Proteus, Pseudomonas, Streptococcus, andChlamydia, and fungal infections such as candidiasis, aspergillosis,histoplasmosis, and cryptococcal meningitis.

In certain embodiments, the pharmaceutical compositions provided hereinare used in the treatment of respiratory diseases and/or disorders,dermatological disorders, ocular diseases and/or disorders,genitourinary diseases and/or disorders including, allograft rejection,auto-immune and allergic, cancer, or damaged or ageing skin such asscarring and wrinkles.

In another aspect, the invention provides a method for generating orpotentiating an immune response in a subject in need thereof, such as amammal, comprising administering an effective amount of a composition asdisclosed herein. The immune response is preferably protective andpreferably involves antibodies and/or cell-mediated immunity. The methodmay be used to induce a primary immune response and/or to boost animmune response.

In certain embodiments, the compositions disclosed herein may be used asa medicament, e.g., for use in raising or enhancing an immune responsein a subject in need thereof, such as a mammal.

In certain embodiments, the compositions disclosed herein may be used inthe manufacture of a medicament for generating or potentiating an immuneresponse in a subject in need thereof, such as a mammal.

The invention also provides a delivery device pre-filled with acomposition or a vaccine disclosed herein.

The mammal is preferably a human, but may be, e.g., a cow, a pig, achicken, a cat or a dog, as the pathogens covered herein may beproblematic across a wide range of species. Where the vaccine is forprophylactic use, the human is preferably a child (e.g., a toddler orinfant), a teenager, or an adult; where the vaccine is for therapeuticuse, the human is preferably a teenager or an adult. A vaccine intendedfor children may also be administered to adults, e.g., to assess safety,dosage, immunogenicity, etc.

One way of checking efficacy of therapeutic treatment involvesmonitoring pathogen infection after administration of the compositionsor vaccines disclosed herein. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses,systemically (such as monitoring the level of IgG1 and IgG2a production)and/or mucosally (such as monitoring the level of IgA production),against the antigen. Typically, antigen-specific serum antibodyresponses are determined post-immunization but pre-challenge whereasantigen-specific mucosal antibody responses are determinedpost-immunization and post-challenge.

Another way of assessing the immunogenicity of the compositions orvaccines disclosed herein where the nucleic acid molecule (e.g., theRNA) encodes a protein antigen is to express the protein antigenrecombinantly for screening patient sera or mucosal secretions byimmunoblot and/or microarrays. A positive reaction between the proteinand the patient sample indicates that the patient has mounted an immuneresponse to the protein in question. This method may also be used toidentify immunodominant antigens and/or epitopes within proteinantigens.

The efficacy of the compositions can also be determined in vivo bychallenging appropriate animal models of the pathogen of interestinfection.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be used to treatboth children and adults. Thus a human subject may be less than 1 yearold, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55years old. Preferred subjects for receiving the compositions are theelderly (e.g., >50 years old, >60 years old, and preferably >65 years),the young (e.g., <5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The compositions are notsuitable solely for these groups, however, and may be used moregenerally in a population.

The compositions disclosed herein that include one or more antigens orare used in conjunction with one or more antigens may be administered topatients at substantially the same time as (e.g., during the samemedical consultation or visit to a healthcare professional orvaccination centre) other vaccines, e.g., at substantially the same timeas a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine,a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzaetype b vaccine, an inactivated poliovirus vaccine, a hepatitis B virusvaccine, a meningococcal conjugate vaccine (such as a tetravalent A CW135 Y vaccine), a respiratory syncytial virus vaccine, etc.

In certain embodiments, the compositions provided herein include oroptionally include one or more immunoregulatory agents such asadjuvants. Exemplary adjuvants include, but are not limited to, a TH1adjuvant and/or a TH2 adjuvant, further discussed below. In certainembodiments, the adjuvants used in the immunogenic compositions provideherein include, but are not limited to:

1. Mineral-Containing Compositions;

2. Oil Emulsions;

3. Saponin Formulations;

4. Virosomes and Virus-Like Particles;

5. Bacterial or Microbial Derivatives;

6. Bioadhesives and Mucoadhesives;

7. Liposomes;

8. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations;

9. Polyphosphazene (PCPP);

10. Muramyl Peptides;

11. Imidazoquinolone Compounds;

12. Thiosemicarbazone Compounds;

13. Tryptanthrin Compounds;

14. Human Immunomodulators;

15. Lipopeptides;

16. Benzonaphthyridines;

17. Microparticles

18. Immunostimulatory polynucleotide (such as RNA or DNA; e.g.,CpG-containing oligonucleotides)

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Cationic Oil-in-Water Emulsions

Squalene, sorbitan trioleate (Span 85), polyoxy-ethylene sorbitanmonololeate (Tween 80) were obtained from Sigma (St. Louis, Mo., USA).1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased fromLipoid (Ludwigshafen Germany).

The components of the emulsions used in these studies are shown in Table1.

TABLE 1 Cationic mg/ml + Squa- CNE Lipid (+) Lipid Surfactant leneBuffer/water CNE17 DOTAP 1.40 0.5% SPAN 85 4.3% 10 mM citrate (in DCM)0.5% Tween 80 buffer pH 6.5 CMF32 DOTAP 3.2 0.5% SPAN 85 4.3% 10 mMcitrate 0.5% Tween 80 buffer pH 6.5 CMF34 DOTAP 4.4 0.5% SPAN 85 4.3% 10mM citrate 0.5% Tween 80 buffer pH 6.5 CMF35 DOTAP 5.0 0.5% SPAN 85 4.3%10 mM citrate 0.5% Tween 80 buffer pH 6.5

CNEs were prepared similar to charged MF59 as previously described (Ottet al., Journal of Controlled Release, volume 79, pages 1-5, 2002). CMF32, 34 and 35 were prepared with one major modification of that process.DOTAP was dissolved in the squalene directly, and no organic solvent wasused. It was discovered that inclusion of a solvent in emulsions thatcontained greater than 1.6 mg/ml DOTAP produced a foamy feedstock thatcould not be microfluidized to produce an emulsion. Heating squalene to37° C. allowed DOTAP to be directly dissolved in squalene, and then theoil phase could be successfully dispersed in the aqueous phase (e.g., byhomogenization) to produce an emulsion. DOTAP is soluble in squalene andhigher concentrations of DOTAP in squalene than those listed in Table 1may be achieved. However, it has been reported that high dose of DOTAPcan have toxic effects. See, e.g., Lappalainen et al., Pharm. Res., vol.11(8):1127-31 (1994).

Briefly, squalene was heated to 37° C., and DOTAP was dissolved directlyin squalene in the presence of SPAN 85. The resulting oil phase was thencombined with the aqueous phase (Tween 80 in citrate buffer) andimmediately homogenized for 2 min using an IKA T25 homogenizer at 24KRPM to produce a homogeneous feedstock (primary emulsions). The primaryemulsions were passed three to five times through a M-110SMicrofluidizer or a M-110P Microfluidizer (Microfluidics, Newton, Mass.)with an ice bath cooling coil at a homogenization pressure ofapproximately 15K-20K PSI. The 20 ml batch samples were removed from theunit and stored at 4° C.

It should be noted that the concentrations of the components of theCNEs, as described in Table 1, are concentrations calculated accordingthe initial amounts of these components that were used to prepare theemulsions. It is understood that during the process of producingemulsions, or during the filter sterilization process, small amounts ofsqualene, DOTAP, or other components may be lost, and the actualconcentrations of these components in the final product (e.g., apackaged, sterilized emulsion that is ready for administration) might beslightly lower, sometimes by up to about 20%. Sometimes, the actualconcentrations of the components in the final product might be slightlylower by up to about 25%, or up to about 35%. However, the conventionalpractice in the art is to describe the concentration of a particularcomponent based on the initial amount that is used to prepare theemulsion, instead of the actual concentration in the final product.

RNA Synthesis

Plasmid DNA encoding an alphavirus replicon (self-replicating RNA) wasused as a template for synthesis of RNA in vitro. Each replicon containsthe genetic elements required for RNA replication but lacks sequencesencoding gene products that are necessary for particle assembly. Thestructural genes of the alphavirus genome were replaced by sequencesencoding a heterologous protein (whose expression is driven by thealphavirus subgenomic promoter). Upon delivery of the replicons toeukaryotic cells, the positive-stranded RNA is translated to producefour non-structural proteins, which together replicate the genomic RNAand transcribe abundant subgenomic mRNAs encoding the heterologousprotein. Due to the lack of expression of the alphavirus structuralproteins, replicons are incapable of generating infectious particles. Abacteriophage T7 promoter is located upstream of the alphavirus cDNA tofacilitate the synthesis of the replicon RNA in vitro, and the hepatitisdelta virus (HDV) ribozyme located immediately downstream of thepoly(A)-tail generates the correct 3′-end through its self-cleavingactivity.

Following linearization of the plasmid DNA downstream of the HDVribozyme with a suitable restriction endonuclease, run-off transcriptswere synthesized in vitro using T7 or SP6 bacteriophage derivedDNA-dependent RNA polymerase. Transcriptions were performed for 2 hoursat 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNApolymerase) final concentration of each of the nucleoside triphosphates(ATP, CTP, GTP and UTP) following the instructions provided by themanufacturer (Ambion, Austin, Tex.). Following transcription, thetemplate DNA was digested with TURBO DNase (Ambion, Austin, Tex.). Thereplicon RNA was precipitated with LiCl and reconstituted innuclease-free water. Uncapped RNA was capped post-transcriptionally withVaccinia Capping Enzyme (VCE) using the ScriptCap m⁷G Capping System(Epicentre Biotechnologies, Madison, Wis.) as outlined in the usermanual. Post-transcriptionally capped RNA was precipitated with LiCl andreconstituted in nuclease-free water. Alternatively, replicons may becapped by supplementing the transcription reactions with 6 mM (for T7RNA polymerase) or 4 mM (for SP6 RNA polymerase) m⁷G(5′)ppp(5′)G, anonreversible cap structure analog (New England Biolabs, Beverly, Mass.)and lowering the concentration of guanosine triphosphate to 1.5 mM (forT7 RNA polymerase) or 1 mM (for SP6 RNA polymerase). The transcripts maybe then purified by TURBO DNase (Ambion, Austin, Tex.) digestionfollowed by LiCl precipitation and a wash in 75% ethanol.

The concentration of the RNA samples was determined by measuring theoptical density at 260 nm. Integrity of the in vitro transcripts wasconfirmed by denaturing agarose gel electrophoresis for the presence ofthe full length construct.

RNA Complexation

The number of nitrogens in solution was calculated from the cationiclipid concentration, DOTAP for example has 1 nitrogen that can beprotonated per molecule. The RNA concentration was used to calculate theamount of phosphate in solution using an estimate of 3 nmols ofphosphate per microgram of RNA. By varying the amount of RNA:Lipid, theN/P ratio can be modified. RNA was complexed to the CNEs in a range ofnitrogen/phosphate ratios (N/P). Calculation of the N/P ratio was doneby calculating the number of moles of protonatable nitrogens in theemulsion per milliliter. To calculate the number of phosphates, aconstant of 3 nmols of phosphate per microgram of RNA was used. Afterthe values were determined, the appropriate ratio of the emulsion wasadded to the RNA. Using these values, the RNA was diluted to theappropriate concentration and added directly into an equal volume ofemulsion while vortexing lightly. The solution was allowed to sit atroom temperature for approximately 2 hours. Once complexed the resultingsolution was diluted to the appropriate concentration and used within 1hour.

Particle Size Assay

Particle size of the emulsion was measured using a Zetasizer Nano ZS(Malvern Instruments, Worcestershire, UK) according to themanufacturer's instructions. Particle sizes are reported as theZ-Average (ZAve) with the polydispersity index (pdi). All samples werediluted in water prior to measurements. Additionally, particle size ofthe emulsion was measured using Horiba LA-930 particle sizer (HoribaScientific, USA). Samples were diluted in water prior to measurements.Zeta potential was measured using Zetasizer Nano ZS using dilutedsamples according to the manufacturer's instructions.

Secreted Alkaline Phosphatase (SEAP) Assay

To assess the kinetics and amount of antigen production, an RNA repliconencoding for SEAP was administered with and without formulation to miceintramuscularly. Groups of 3 or 5 female Balb/C mice aged 8-10 weeks andweighing about 20 g were immunized with CNEs complexed with replicon RNAencoding for SEAP. Naked RNA was formulated in RNase free 1×PBS. A 100μl dose was administered to each mouse (50 μl per site) in thequadriceps muscle. Blood samples were taken 1, 3, and 6 days postinjection. Serum was separated from the blood immediately aftercollection, and stored at −30° C. until use.

A chemiluminescent SEAP assay Phospha-Light System (Applied Biosystems,Bedford, Mass.) was used to analyze the serum. Mouse sera was diluted1:4 in 1× Phospha-Light dilution buffer. Samples were placed in a waterbath sealed with aluminum sealing foil and heat inactivated for 30minutes at 65° C. After cooling on ice for 3 minutes, and equilibratingto room temperature, 50 uL of Phospha-Light assay buffer was added tothe wells and the samples were left at room temperature for 5 minutes.Then, 50 uL of reaction buffer containing 1:20 CSPD® (chemiluminecentalkaline phosphate substrate) substrate was added, and the luminescencewas measured after 20 minutes of incubation at room temperature.Luminescence was measured on a Berthold Centro LB 960 luminometer (OakRidge, Tenn.) with a 1 second integration per well. The activity of SEAPin each sample was measured in duplicate and the mean of these twomeasurements is shown.

Example 2: The Effect of Particle Size on Immunogenicity

This example shows that particle size affects the immunogenicity of theCNE/RNA formulations.

A. Protocols for particle size assay and in vivo SEAP assay aredescribed in Example 1. Protocols for murine immunogenicity studies aredescribed in Example 3. FIG. 1A shows the results (arithmetic mean) ofthe in vivo SEAP assay. FIG. 1B shows the total IgG titers of individualanimals in the BALB/c mice at 2wp1 and 2wp2 time points.

RNA complexation with CNE17 increased the size of the emulsion particlesfrom about 220 nm to about 300 nm. As shown in FIG. 1A and FIG. 1B, asparticle size increased, the expression levels of SEAP were reduced, andthe host immune responses were also decreased.

B. CMF34 prepared at different sizes was complexed with RNA encodingRSV-F at a 7:1 theoretical N/P ratio and injected into the quadricepsmuscle of Balb/C mice bilaterally (0.05 ml/site). Emulsion particlesizes were modulated by increasing the processing pressure of themicrofluidizer. The data (Table 2) shows the highest anti-RSV F titerswere generated when the emulsion particle size was smaller (see 120 nmand 90 nm particles) at two weeks after the first immunization.

TABLE 2 RNA N/P 2wp1 2wp2 Formulation dose ratio GMT GMT 1 ug vA375batch 1 1 ug 7:1 16 151 1 ug vA375 batch 1 + CMF34 (5k PSI 1 ug 7:1 1621644 processing - 200 nm size) 1 ug vA375 batch 1 + CMF34 (7k PSI 1 ug7:1 183 2540 processing - 150 nm size) 1 ug vA375 batch 1 + CMF34 (12kPSI 1 ug 7:1 465 3563 processing - 120 nm size) 1 ug vA375 batch 1 +CMF34 (20k PSI 1 ug 7:1 548 2542 processing - 90 nm size)

Example 3: Effects of Buffer Compositions on Immunogenicity and ParticleSize

In this example, various emulsions based on CNE17 but with differentbuffer components were prepared. Table 3 shows the compositions of thebuffer-modified emulsions.

TABLE 3 Base Emulsion Buffer/water CNE17: 4.3% Squalene, 0.5% SPAN 85, 0mM citrate buffer 0.5% Tween 80, 1.4 mg/ml DOTAP (in RNase-free dH₂O, noDCM) CNE17: 4.3% Squalene, 0.5% SPAN 85, 1 mM citrate buffer 0.5% Tween80, 1.4 mg/ml DOTAP (in RNase-free dH₂O, no DCM) CNE17: 4.3% Squalene,0.5% SPAN 85, 5 mM citrate buffer 0.5% Tween 80, 1.4 mg/ml DOTAP (inRNase-free dH₂O, no DCM) CNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mMcitrate buffer pH 0.5% Tween 80, 1.4 mg/ml DOTAP 6.5 300 mM TrehaloseCNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mM citrate buffer pH 0.5% Tween80, 1.4 mg/ml DOTAP 6.5 300 mM Sucrose CNE17: 4.3% Squalene, 0.5% SPAN85, 10 mM citrate buffer pH 0.5% Tween 80, 1.4 mg/ml DOTAP 6.5 300 mMSorbitol CNE17: 4.3% Squalene, 0.5% SPAN 85, 10 mM citrate buffer pH0.5% Tween 80, 1.4 mg/ml DOTAP 6.5 300 mM Dextrose

In vitro binding assay showed that reducing the concentration of citratebuffer caused RNA to bind more tightly.

Results from murine immunogenicity studies showed that adding sugars toCNE17 did not significantly impact the immunogenicity of theCNE17-formulated RNA (Table 4, groups 9-12)). Slight increases in IgGtiters were observed with the addition of sorbitol and dextrose.

TABLE 4 Description N:P 2wp2/2wp1 Group # Emulsion ratio 2wp1 2wp2 ratio1 1 ug vA317 — 77 1,710 22.2 2 RV01(15) — 3,441 59,557 17.3 3 CNE17DOTAP 10:1 1,474 6,512 4.4 4 CNE13 DDA 18:1 482 8,385 17.4 5 CMF37 DOTMA10:1 474 6,556 13.8 6 CNE16 DOEPC 12:1 1,145 9,673 8.4 7 CMF42 DSTAP10:1 22 148 6.7 8 DDA Liposomes 18:1 898 5,333 5.9 9 CNE17 with 300 mM10:1 1,807 6,445 3.6 Trehalose 10 CNE17 with 300 mM 10:1 1,042 5,515 5.3Sucrose 11 CNE17 with 300 mM 10:1 1,209 8,874 7.3 Sorbitol 12 CNE17 with300 mM 10:1 1,247 7,956 6.4 Dextrose Groups 1-8 had 5 animals/group, andgroups 9-12 had 10 animals/group.

Table 5 summarizes the results of murine immunogenicity studies whenCNE17-formulated RNAs were prepared using different buffer systems.

TABLE 5 2wp2/ Description 2wp1 Group # RNA Emulsion N:P ratio 2wp1 2wp2ratio 1 1 μg RSV-F* PBS — 100 2269 23 2 RV01(15) PBS — 8388 105949 13 31 μg RSV-F* CNE17 with 280 mM Sucrose 10:1 898 9384 10 4 1 μg RSV-F*CNE17 with 280 mM sucrose, 10:1 1032 3184 3.1 10 mM NaCl, 1 mM Citrate,5 CNE17 with 280 mM sucrose, 10:1 79 895 11.3 10 mM NaCl, 1 mM Citrate,0.5% (w/v) and Pluronic F127 *vA375 replicon, **vA317 replicon.Replicons were Ambion transcribed in HEPES buffer, then (i) LiClprecipitated, (ii) capped in Tris buffer, and (iii) LiCl precipitated.All groups had 8 animals/group.

Different buffer compositions also affected particle size. As shown inFIG. 2A, addition of sugar (sucrose) decreased the particle size of theRNA/CNE complex; addition of low concentrations of NaCl (at 10 mM) alsodecreased the particle size of the RNA/CNE complex (FIG. 2A). Citratebuffer did not affect the particle size of the RNA/CNE complex (FIG.2B).

The effects of polymers on particle size are shown in FIG. 2C. Inparticular, addition of 0.5% pleuronic F127 to RNA buffer reduced theparticle size of the RNA/CNE complex to the pre-complexation size (CNEparticles without RNA).

The total antibody titers and neutralizing antibody titers of CNE17 inpreferred buffer systems, 280 mM sucrose, 10 mM NaCl, and 1 mM Citrate;or 280 mM sucrose, 10 mM NaCl, 1 mM Citrate, and 0.5% (w/v) PluronicF127, are shown in Table 5 (groups 4 and 5).

Example 4: Effects of N/P Ratio on Immunogenicity

In this example, the RNA replicon vA375, which encodes an RSV F antigen,was formulated as liposome (RV01), complexed with CNE17 at N/P of 10:1,and with CMF32 or CMF34 at theoretical N/P of 12;1, 10:1, 8:1, 6:1, and4:1. Theoretical N/P ratios reflect the N/P ratios calculated accordingto the initial amounts of DOTAP and RNA that were used to prepare theformulations. Actual N/P ratios were slightly lower than theoretical N/Pratios because small amounts of DOTAP were lost during preparation ofthe emulsions. The GMT data reflect the mean log₁₀ titer of individualmice in each group (8 mice/group). All formulations were adjusted to 300mOsm/kg with sucrose prior to immunization. There were no obvioustolerability issues observed (e.g., body weight, early serum cytokines)with either CMF32 or CMF34 formulations.

Actual N/P ratios were determined by quantifying DOTAP content in CNE orCMF batches using HPLC with a charged aerosol detector (Corona Ultra,Chelmsford, Mass.). The CNE and CMF samples were diluted in isopropanoland injected onto a XTera C18 4.6×150 mm 3.5 um column (Waters, Milford,Mass.). The area under the curve was taken from the DOTAP peak in thechromatogram and the concentration was interpolated off a DOTAP standardcurve. Using the actual DOTAP concentration, an actual N/P ratio was becalculated.

The immunogenicity data in Table 6 show that good titers were obtainedwhen the actual N/P ratio was at least 4:1.

TABLE 6 RNA Actual 2wp2/ Form- (μg/ Theoretical N/P 2wp1 2wp2 2wp1ulation dose) N/P ratio ratio GMT GMT (boost) Naked 1 — — 68 1019 15RV01 1 — — 9883 68116 7 CNE17 1 10:1 — 1496 6422 4 CMF32 1 12:1 9.4:12617 14246 5 1 10:1 (batch 1) 6.0:1 1537 10575 7 1 10:1 (batch 2) 8.0:12047 16244 8 1  8:1 6.3:1 2669 7656 3 1  6:1 4.7:1 1713 4715 3 1  4:13.1:1 872 3773 4 CMF34 1 12:1 7.4:1 3141 10134 3 1 10:1 (batch 1) 6.1:11906 11081 6 1 10:1 (batch 2) 7.0:1 2388 9857 4 1  8:1   5:1 1913 8180 41  6:1 3.7:1 1764 6209 4 1  4:1 2.5:1 1148 4936 4Sequences

A317 (SEQ ID NO:1)

A375 (SEQ ID NO:2)

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

The invention claimed is:
 1. A method for generating an immune responsein a subject, comprising administering to the subject an effectiveamount of an immunogenic cationic oil-in-water emulsion comprisingemulsion particles that contain an oil core and a cationic lipid, and anucleic acid molecule that is complexed to the emulsion particles,wherein: a) the nucleic acid is RNA that encodes a polypeptide antigen,b) the average diameter of the emulsion particles is from about 80 nm toabout 180 nm, and c) the N/P of the emulsion is at least 4:1; with theproviso that the nucleic acid molecule does not encode secreted alkalinephosphatase, and the further proviso that the nucleic acid molecule isnot the RNA encoded by the plasmid A317, the sequence of which is givenby SEQ ID NO:1; whereby the immune response is generated in the subject.2. The method of claim 1, wherein the RNA is self-replicating RNA. 3.The method of claim 1, wherein the immunogenic cationic oil-in-wateremulsion is buffered, and has a pH from about 6.0 to about 8.0.
 4. Themethod of claim 3, wherein the immunogenic cationic oil-in-wateremulsion comprises a buffer selected from the group consisting of acitrate buffer, a succinate buffer, an acetate buffer, and combinationsthereof.
 5. The method of claim 4, wherein the buffer is a citratebuffer and the pH is about 6.5.
 6. The method of claim 1, furthercomprising an inorganic salt.
 7. The method of claim 6, wherein theinorganic salt concentration is no greater than 30 mM.
 8. The method ofclaim 1, further comprising a nonionic tonicifying agent.
 9. The methodof claim 8, wherein the nonionic tonicifying agent is a sugar, a sugaralcohol or combinations thereof.
 10. The method of claim 9, wherein thenonionic tonicifying agent is selected from the group consisting ofsucrose, trehalose, sorbitol, dextrose and combinations thereof.
 11. Themethod of claim 1, further comprising a polymer in the aqueous phase.12. The method of claim 11, wherein the polymer is a poloxamer.
 13. Themethod of claim 11, wherein the polymer is Pluronic F127.
 14. The methodof claim 11, wherein the emulsion contains about 0.05% to about 20%(w/v) polymer.
 15. The method of claim 1, wherein the average diameterof the emulsion particles is from about 80 nm to about 130 nm.
 16. Themethod of claim 1, wherein the N/P of the emulsion is from 4:1 to about20:1.
 17. The method of claim 1, wherein the N/P of the emulsion is from4:1 to about 15:1.
 18. The method of claim 1, wherein the cationicoil-in-water emulsion is isotonic with human blood.
 19. The method ofclaim 1, wherein the oil core comprises an oil that is selected from thegroup consisting of: Castor oil, Coconut oil, Corn oil, Cottonseed oil,Evening primrose oil, Fish oil, Jojoba oil, Lard oil, Linseed oil, Oliveoil, Peanut oil, Safflower oil, Sesame oil, Soybean oil, Squalene,Squalane, Sunflower oil, Wheatgerm oil, and combinations thereof. 20.The method of claim 19, wherein the oil is Squalene.
 21. The method ofclaim 1, wherein the cationic lipid is selected from the groupconsisting of: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP),3β-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DCCholesterol), dimethyldioctadecylammonium (DDA),1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP),dipalmitoyl(C_(16:0))trimethyl ammonium propane (DPTAP),distearoyltrimethylammonium propane (DSTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), Lipids E0001-E0118,and combinations thereof.
 22. The method of claim 21, wherein thecationic lipid is DOTAP.
 23. The method of claim 1, wherein the cationiclipid comprises a quaternary amine.
 24. The method of claim 1, whereinthe particle further comprises a surfactant.
 25. The method of claim 24,wherein the surfactant is a nonionic surfactant.
 26. The method of claim25, wherein the surfactant is Sorbitan Trioleate, polysorbate 80, or acombination thereof.
 27. The method of claim 1, wherein the emulsionfurther comprises an antioxidant.